![Chapter 1
High-Throughput Bioluminescence Imaging and Reporter
Gene Assay with 3D Spheroids from Human Cell Lines
Maria Maddalena Calabretta, Laura Montali, Antonia Lopreside,
Elisa Michelini, and Aldo Roda
Abstract
3D cell culture models represent an attractive approach to decode intracellular and intercellular signaling,
providing biologically relevant information and predictive data. Bioluminescent reporter gene assays and
bioluminescence imaging in 3D cell models are very promising bioanalytical tools for several applications.
Here we report a very straightforward method for bioluminescence imaging and bioluminescent reporter
gene assays in 3D cell-culture models. Both the assays can be easily implemented in laboratories equipped
with basic cell culture facilities and instrumentation for bioluminescence detection, that is, low-light
detectors connected to inverted microscopes and luminometers, without the need for additional
equipment.
Key words Bioluminescence, Spheroids, Imaging, Reporter gene, 3D cell models, Luciferase
1 Introduction
Bioluminescent (BL) cell-based assays based on two-dimensional
(2D) cell cultures represent a well-established bioanalytical tool for
interrogating natural biological phenomena, including gene
expression, promoter regulation, and for monitoring molecular
pathways, thus providing highly valuable information. Major appli-
cations of these assays are in drug discovery but also span from
regenerative medicine to environmental monitoring and food con-
trol, as well as forensic applications [1–3]. Several cell-based assays
have been developed based on bioluminescent reporters such as
luciferases. Reporter gene technology relies on the splicing of
transcriptional control elements to a variety of reporter proteins
to monitor cellular events associated with gene expression and
intracellular signaling cascades. The main advantages of these assays
are wide dynamic range, high sensitivity, and suitability to high
throughput-screening. Moreover, cell-based assays relying on luci-
ferases as reporter protein are generally very simple to implement
Steven Ripp (ed.), Bioluminescent Imaging: Methods and Protocols, Methods in Molecular Biology, vol. 2081,
https://doi.org/10.1007/978-1-4939-9940-8_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020
3](https://image.slidesharecdn.com/23858053-250523044023-b9fb45dd/85/Bioluminescent-Imaging-Methods-And-Protocols-1st-Steven-Ripp-17-320.jpg)
![and do not require expensive equipment or skilled personnel. More
recently, thanks to the availability of luciferases emitting at different
colors or requiring different substrates, multiplexed assays have also
been developed to monitor two or more analytes or to include
viability controls to correct the analytical signal according to cell
viability [4, 5]. The recent technical advancements have dramati-
cally reshaped the way cell-based assays are developed, expanding
their applicability outside laboratories for on-site analyses. Several
whole-cell biosensors have been reported in the past years, relying
on the use of portable light detectors (such as charge-coupled
devices and complementary metal oxide semiconductors) or simply
integrated into 3D-printed smartphone-based devices [6–8]. These
approaches are surely promising; however, 2D cell cultures do not
always resemble the morphology and the functionality of the natu-
ral 3D environment. As a result, 2D cell culture tests can provide
misleading data, reducing the reliability and predictivity of the
results. Instead, 3D cell models are characterized by complex cell-
to-cell communication and formation of extracellular matrix
(ECM), thus restoring complex tissue architecture [9].
3D cell culture models represent an attractive approach to
decode intra- and intercellular signaling, providing biologically
relevant information and predictive data [10]. It is known that
cells in 3D culture environments differ in gene, protein, and cell
receptor expression from 2D-monolayer cell cultures and they
provide an excellent model as surrogate in vivo systems. 3D struc-
ture spheroids exhibit enhanced cell viability, stable morphology
and polarization, increasing proliferative activity and physiological
metabolic function, which are markedly improved when compared
to 2D cell monolayers [11, 12].
Besides the European Union, the USA and many other
countries are strongly encouraging the implementation of in vitro
assays and other approaches to replace animal testing, in accordance
with the “three Rs principle” of Russell and Burch [13]. The
possibility of exploiting bioluminescence detection for monitoring
3D cell models has been previously demonstrated by us and others
[14–17].
We have previously reported a high-throughput BL 3D cell-
based assay in micropatterned 96-well plate format using the tran-
scriptional regulation of nuclear factor K beta response element in
human embryonic kidney (HEK293) cells. Here we report a very
straightforward method for bioluminescence imaging and biolumi-
nescent reporter gene assays in 3D cell-culture models. Both the
assays can be easily implemented in laboratories equipped with
basic cell culture facilities and instrumentation for bioluminescence
detection, that is, luminometer and low-light detectors connected
to inverted microscopes, without the need for additional
equipment.
4 Maria Maddalena Calabretta et al.](https://image.slidesharecdn.com/23858053-250523044023-b9fb45dd/85/Bioluminescent-Imaging-Methods-And-Protocols-1st-Steven-Ripp-18-320.jpg)
![2 Materials
2.1 3D Cell Culture
and Transfection
1. Human embryonic kidney HEK293 cell line (ATCC®
CRL-1573) or other cell lines that can be transfected readily.
2. Growth Medium: Dulbecco’s modified Eagle’s medium
(DMEM) high glucose. Supplemented with 10% fetal bovine
serum (FBS), 2 mM L-glutamine, 50 μg/mL penicillin and
50 μg/mL streptomycin (Carlo Erba reagents, Cornaredo,
MI, Italy). Filter the growth medium using a filter cup
0.22 μm. Store at 4
C (see Note 1).
3. Trypsin–EDTA solution 1 (Carlo Erba reagents, Cornaredo,
MI, Italy). Store at 4
C.
4. T25 flask culture and serological pipettes.
5. 96-well microspace round bottom cell culture plates (Elpla-
sia™, Kuraray, Japan).
6. FuGENE HD transfection reagent (Promega, Madison, WI).
7. Plasmids: pGL4.32[PLG2] obtained by replacing Luc2P from
vector pGL4.32[luc2P/NF-kB-RE/Hygro] (Promega, Madi-
son, WI) with PLG2 luciferase gene [18]; plasmid for constitu-
tive expression of the luciferase (e.g., pCMV_PLG2) to
characterize luciferase emission in 3D cell cultures.
2.2 Reagents 1. 0.1 M phosphate buffered saline (PBS): Add 8 g of NaCl,
200 mg of KCl, 1.44 g of Na2HPO4, 240 mg of KH2PO4
into 1 L of double-distilled water. Adjust solution to desired
pH (typically pH 7.4). Sterilize the solution in autoclave and
prepare 50 mL aliquots. Store at room temperature.
2. 1.0 mM D-luciferin solution: Prepare Buffer n.1 (0.1 M citric
acid solution) by dissolving 21.0 g of citric acid monohydrate,
C6H8O7·H2O into 1 L of distilled water; prepare Buffer n.2
(0.1 M trisodium citrate solution) by dissolving 29.41 g of
trisodium citrate dihydrate, C6H5O7Na3·2H2O, into 1 L of
distilled water. Mix 35 mL of Buffer n.1 and 65 mL Buffer n.2
for 30 min to obtain citric acid-sodium citrate buffer solution
at pH 5.0. Dissolve 28.3 mg of D-luciferin into 100 mL of citric
acid–sodium citrate buffer solution at pH 5.0. Prepare
8–10 mL aliquots and store at 20
C (see Note 2).
3. Tumor Necrosis Factor Alpha (TNFα) stock solution: Centri-
fuge the vial before opening. Reconstitute the TNFα with 1 mL
PBS sterile solution 0.1 M pH 7.4 containing 0.1% endotoxin-
free recombinant human serum albumin in order to obtain a
stock solution 10.0 μg/mL. Prepare 100 μL aliquots and store
at 20
C (see Note 3). Prepare on the day of the assay serial
dilutions of TNFα (concentration range 0.1–50.0 ng/mL)
using sterile deionized water.
Bioluminescence Imaging of 3D Spheroids 5](https://image.slidesharecdn.com/23858053-250523044023-b9fb45dd/85/Bioluminescent-Imaging-Methods-And-Protocols-1st-Steven-Ripp-19-320.jpg)
![6. Remove the supernatant and add 5 mL of prewarmed complete
growth medium, resuspend cells by gently pipetting.
7. Count the cells and plate them in a 96-well microspace round
bottom Elplasia cell culture plate at a concentration of 2 104
cells per well in a total volume of 200 μL of medium (see
Note 6).
8. Place the plate in incubator at 37
C with 5% CO2 for 48–72 h
for spheroid formation (see Note 7).
3.2 Spheroid
Analysis
1. Analyze bright-field images of HEK293 spheroids at desired
time points (e.g., every 24 h) using ImageJ version 1.51d
software to calculate the projected area (A) and perimeter (P)
of each spheroid according to [19]. Calculate a sphericity factor
(ϕ) as follows:
ϕ ¼
π
ffiffiffiffiffi
4A
π
q
P
2. Determine the projected area and perimeter for each spheroid
using ImageJ and export data to MS EXCEL to calculate the
sphericity factor of about 30 spheroids to obtain an average
value standard deviation (see Note 8).
3. Calculate the average diameter of 30 spheroids, generally after
72 h of incubation (cell density of 4 104
cells) HEK293
spheroids with an average diameter of 210 25 μm can be
obtained (see Note 9).
3.3 Spheroid
Transfection
Transfect 3-day-old HEK293 spheroids with 0.10 μg of plasmid
per well of a 96-well Elplasia plate with Fugene®
HD reagent with a
ratio of 3:1 (see Note 10). Add a total volume of transfection mix
(plasmid DNA and FuGENE®
HD) of 2–10 μL per well. For
transfecting 10 wells prepare the following mixture.
1. Mix by inverting briefly the Fugene HD reagent (see Note 11).
2. Add sterile deionized water to a sterile polystyrene Eppendorf
to reach 62 μL final volume.
3. Add 1.1 μg plasmid in sterile H2O and mix immediately by
pipetting.
4. Add 3.3 μL Fugene HD reagent (ratio 3:1) and mix briefly by
vortexing.
5. Incubate the Fugene HD reagent/DNA mixture for 5–10 min
at room temperature.
6. Add 5 μL of this mixture per well and mix by gently moving the
plate (see Note 12).
7. Place the plate in incubator at 37
C, 5% CO2 for 48 h.
Bioluminescence Imaging of 3D Spheroids 7](https://image.slidesharecdn.com/23858053-250523044023-b9fb45dd/85/Bioluminescent-Imaging-Methods-And-Protocols-1st-Steven-Ripp-21-320.jpg)
![Chapter 2
Bioluminescence Resonance Energy Transfer (BRET)
Coupled Near-Infrared Imaging of Apoptotic Cells
Setsuko Tsuboi and Takashi Jin
Abstract
Detection of apoptotic cells is crucial for understanding the mechanism of diseases and for therapy
development. So far, visible-emitting fluorescent probes such as FITC-labeled Annexin V has been widely
used for the detection of apoptotic cells. However, such probes cannot be applied to noninvasive imaging in
the near-infrared (NIR) region. Compared with visible light, NIR light is highly permeable in turbid
biological samples and tissues. In addition, NIR optical imaging has several advantages such as lower
autofluorescence and scattering from biological samples, leading to clearer images with high signal to
background ratios. Here, we describe the synthesis and application of bioluminescence resonance energy
transfer (BRET)-coupled quantum dots (QDs) for the NIR optical imaging of apoptotic cells.
Key words Bioluminescence resonance energy transfer (BRET), Renilla luciferase, Near-infrared
imaging (NIR imaging), Quantum dots (QDs), Annexin V, Apoptotic cell
1 Introduction
Programmed cell death (apoptosis) plays an important role not only
in the homeostasis of normal tissues but also in the pathological
conditions of diseased tissues [1, 2]. The detection of apoptotic
cells is crucial for understanding the mechanism of those diseases
and for therapy development. Deregulation in apoptosis induces
numerous diseases such as cancer and cardiovascular and neurode-
generative diseases [2, 3]. During the early process of apoptosis [4],
morphological changes of the cell membrane induce the asymmet-
rical distribution of phosphatidylserine (PS) on the outer mono-
layer of the membrane [5, 6]. Annexin V is an endogenous protein
having binding ability to PS in the presence of Ca2+
ions, and this
protein is widely used as a probe for the detection of apoptotic cells
[7]. So far, for the detection of apoptotic cells, Annexin V has been
modified with fluorescence, luminescence, magnetic resonance
contrast, and radioisotopes [7]. Among these probes, fluorescent-
dye labeled probes such as FITC- and Cy3/Cy5-Annexin V [8, 9]
Steven Ripp (ed.), Bioluminescent Imaging: Methods and Protocols, Methods in Molecular Biology, vol. 2081,
https://doi.org/10.1007/978-1-4939-9940-8_2, © Springer Science+Business Media, LLC, part of Springer Nature 2020
15](https://image.slidesharecdn.com/23858053-250523044023-b9fb45dd/85/Bioluminescent-Imaging-Methods-And-Protocols-1st-Steven-Ripp-29-320.jpg)
![are the most popular for the optical detection of apoptotic cells by
using fluorescence microscopy or fluorescence activated cell sort-
ing. However, most fluorescence-labeled Annexin V-probes emit at
the visible region less than 700 nm. Such visible-emitting probes
encounter high background signals arising from autofluorescence
of intrinsic fluorescent species in biological samples.
We have developed a NIR optical imaging technique for the
detection of apoptotic cells using bioluminescence resonance
energy transfer (BRET) [10–12] coupled annexin V functionalized
NIR-QDs [13, 14]. Compared with fluorescence imaging, BRET-
based luminescence imaging does not require external excitations,
leading to low autofluorescence from biological samples. In this
chapter, we describe the synthesis and application of recombinant
Annexin V protein functionalized QDs for BRET-coupled NIR
optical detection of apoptotic cells (Fig. 1). This probe is designed
to emit NIR luminescence due to BRET from Renilla luciferase
(RLuc) [15]-coelenterazine (CTZ) to glutathione (GSH) coated
NIR emitting CdSeTe/CdS QDs (GSH-QDs) [16, 17].
2 Materials
2.1 Synthesis of NIR-
Emitting QDs
1. Selenium (powder, 99.999%).
2. Tellurium (shot, 1–2 mm, 99.99%).
3. Cadmium 2,4-pentanedionate (ODPA).
4. Tri-octylphosphine oxide (TOPO).
5. Trioctylphosphine (TOP).
6. Tri-butylphosphine (TBP).
7. Hexadecylamine (HDA, 90%).
8. Sulfur (S, crystalline, 99.999%).
9. Inert gas (nitrogen).
10. Fluorescence spectrometer.
Fig. 1 Schematic representation for the preparation of a recombinant protein, HisRLuc-Annexin V functiona-
lized QDs (Annexin V·RLuc-QDs), where HisRLuc·Annexin V protein directly binds to the surface of GSH-QDs.
BRET occurs from CTZ to QD in an Annexin V·RLuc-QD
16 Setsuko Tsuboi and Takashi Jin](https://image.slidesharecdn.com/23858053-250523044023-b9fb45dd/85/Bioluminescent-Imaging-Methods-And-Protocols-1st-Steven-Ripp-30-320.jpg)
![2.5 Protein
Conjugation to GSH-
QDs
1. Aqueous solution of HisRLuc-Annexin V (1 mg/mL in PBS).
2. Aqueous solution of GSH-QDs (1 μM in 10 mM Na2CO3,
O.D. ¼ ca. 3 at 488 nm).
3. PBS.
4. Gel filtration column.
2.6 BRET Spectra
of Annexin V·RLuc-QDs
1. Aqueous solution of Annexin V·RLuc-QDs (1 μM in PBS).
2. PBS.
3. Ethanol solution (1 mg/mL) of coelenterazine (CTZ).
4. Luminescence spectrometer.
2.7 Cell Preparation
and Apoptosis
Induction
1. Desired cell line for biologic question of interest.
2. General apparatus and reagents used for cell culture. Medium,
culture dish, incubator, microscope, and so on.
3. Actinomycin D (ActD).
2.8 Flow Cytometric
Analysis and Cell
Imaging
1. 1 Annexin V binding buffer (2.5 mM CaCl2 in medium).
2. FITC-Annexin V.
3. Propidium iodide (PI) solution.
4. DMSO solution of ActD.
5. Flow cytometer.
2.9 BRET-Coupled
Luminescence
Imaging of Apoptotic
Cells
1. Aqueous solution of Annexin V·RLuc-QDs (1 μM in PBS).
2. Ethanol solution (1 mg/mL) of CTZ.
3. Luminescence imaging system.
3 Methods
NIR-emitting CdSeTe/CdS (core/shell) QDs are prepared by a
high temperature decomposition method [16, 17]. A recombinant
Annexin V protein (HisRLuc·Annexin V) was prepared by the
transformation of pRSET-RLuc-Annexin V plasmid into E. coli.
HisRLuc·Annexin V protein has six histidine tags which directly
bind to the surface of GSH capped CdSeTe/CdS QDs (Fig. 1).
This is due to the high affinity of histidine to Cd2+
ions at the
surface of a Cd-S shell layer [18–20]. The GSH capped CdSeTe/
CdS QDs (GSH-QDs) show a broad absorption spectrum from
NIR to visible region (400–800 nm) to be able act as an acceptor
for the CTZ emission (around 485 nm).
18 Setsuko Tsuboi and Takashi Jin](https://image.slidesharecdn.com/23858053-250523044023-b9fb45dd/85/Bioluminescent-Imaging-Methods-And-Protocols-1st-Steven-Ripp-32-320.jpg)
![puberty, reduced sperm counts, and various other adverse health
effects [1, 2]. Unfortunately, many compounds that make up
everyday consumer products such as plastics, household and per-
sonal care products, pharmaceuticals, and pesticides have been
found to be EDCs, and their wide use and discharge into the
environment through anthropogenic activities and industrial
waste has made monitoring of EDCs in the aqueous ecosystem a
critical checkpoint to safeguard the health of humans and wildlife.
Analytical methods such as gas chromatography/mass spec-
trometry are the traditional gold standards for EDC detection.
However, despite their supreme sensitivity, analytical methods are
expensive, time-consuming, require specialized instruments and
personnel, and most importantly, lack the capability to predict
EDC bioavailability. As a complement to analytical methods, a
variety of whole-cell bioassays have been developed to provide a
biological evaluation of the cumulative effects of EDCs in environ-
mental samples without identifying individual compounds. Using
the lower eukaryotic organism Saccharomyces cerevisiae as a plat-
form for EDC screening, two bioluminescent yeast assays BLYES
[3] and BLYAS [4] have been developed to detect estrogenic and
androgenic activity, respectively. These genetically engineered
S. cerevisiae strains chromosomally express a hormone-specific
nuclear receptor (i.e., human estrogen receptor alpha in BLYES
and human androgen receptor in BLYAS), which upon activation
by EDC exposure will turn on the episomal expression of a reporter
operon under the control of a hormone-specific promoter. In
particular, BLYES and BLYAS express the complete Photorhabdus
luminescens luciferase gene cassette (luxCDABE) that allows for
autonomous bioluminescent production in a dose-dependent man-
ner without the need of exogenous substrate addition or photonic
excitation. A constitutively bioluminescent strain BLYR [5] has also
been developed and is often used in parallel with BLYES and/or
BLYES strains to assess the toxicity of environmental samples.
These yeast bioassays can be conveniently performed in test
tubes for low-throughput applications or in microtiter plates (i.e.,
96-well plates) for simultaneous analysis of multiple samples in a
single assay. They provide rapid (usually within a few hours), cost-
effective, and high-throughput detection of EDCs and have been
successfully applied worldwide for chemical screening and evalua-
tion [6–9] and EDC monitoring in surface and drinking water
[10, 11] and wastewater [12, 13]. The bioluminescent signal is
easily detectable by conventional luminometers for
low-throughput applications, or by microplate readers with lumi-
nescence capability for high-throughput assays. Here we present
the detailed protocol of using the BLYES, BLYAS, and BLYR
bioluminescent yeast bioreporters in a 96-well plate format to
analyze EDC contaminants in environmental samples.
30 Tingting Xu et al.](https://image.slidesharecdn.com/23858053-250523044023-b9fb45dd/85/Bioluminescent-Imaging-Methods-And-Protocols-1st-Steven-Ripp-43-320.jpg)
![2 Materials
2.1 Yeast Strains Bioluminescent yeast bioreporter strains used in this protocol
include Saccharomyces cerevisiae BLYES for estrogenicity detection,
BLYAS for androgenicity detection, and BLYR for toxicity evalua-
tion [3, 4, 6].
2.2 Glassware Glassware must be used for medium preparation and storage and
yeast growth. Wrap glassware openings with aluminum foil and
sterilize by baking at 500
C for 4 h in a muffle furnace (see Note 1).
2.3 Chemical
Standards
Serial dilutions of 17β-estradiol (E2) and 5α-dihydrotestosterone
(DHT) are used as positive controls and calculation standards for
BLYES estrogenicity and BLYAS androgenicity detection, respec-
tively. Methanol (HPLC grade) is used as solvent and negative
control in BLYES and BLYAS assays. Chemical standards are
prepared as below.
1. Dissolve 13.67 mg E2 in 5 mL of methanol in a borosilicate
glass vial to make a 1 102
M E2 stock.
2. Serially dilute the 1 102
M E2 stock by tenfold in separate
glass EPA vials with methanol to final concentrations of
1 103
M, 1 104
M, 1 105
M, 1 106
M,
1 107
M, 1 108
M, 1 109
M, 1 1010
M, and
1 1011
M.
3. Dilute the 1 106
M E2 stock by twofold with methanol to
5 107
M.
4. Perform 5 tenfold serial dilutions of the 5 107
M E2
solution in separate glass EPA vials with methanol to final
concentrations of 5 108
M, 5 109
M, 5 1010
M,
5 1011
M, and 5 1012
M.
5. Dilute the 5 107
M E2 solution by twofold with methanol
in a glass vial to 2.5 107
M.
6. Perform 5 tenfold serial dilutions of the 2.5 107
M E2
solution in separate glass EPA vials with methanol to final
concentrations of 2.5 108
M, 2.5 109
M,
2.5 1010
M, 2.5 1011
M, and 2.5 1012
M.
7. The final full series of E2 standards will include 18 dilutions,
ranging from 1 106
M to 2.5 1012
M.
8. To prepare DHT standards, first dissolve 14.52 mg DHT into
5 mL methanol to generate a 1 102
M stock.
9. From the 1 102
M DHT stock, follow similar dilution
patterns in Subheading 2.3 steps 2–6 to generate a full series
of DHT standards consisting of 18 dilutions ranging from
1 105
M to 2.5 1011
M (see Note 2).
10. Store E2 and DHT standards at 4
C.
Monitoring Emerging Contaminants Through Microbial Bioreporters 31](https://image.slidesharecdn.com/23858053-250523044023-b9fb45dd/85/Bioluminescent-Imaging-Methods-And-Protocols-1st-Steven-Ripp-44-320.jpg)
![Table 2
An example data set of BLYES estrogenicity, BLYAS androgenicity, and BLYR toxicity test of
environmental samples
Sample dilutions Concentration factor () BLYR (RLU) BLYES (RLU) BLYAS (RLU)
500 216 342 4133
250 249 209 2639
125 255 191 1514
50 287 170 707
25 295 283 857
12.5 286 265 539
5 328 222 522
2.5 352 198 549
1.25 348 187 559
0.5 337 166 521
0.25 341 168 523
0.125 331 163 501
Methanol control BLYR (RLU) BLYES (RLU) BLYAS (RLU)
347 175 526
340 190 498
341 183 496
Chemical standards [E2] (M) BLYES (RLU) [DHT] (M) BLYAS (RLU)
1 107
3934 1 106
8263
5 108
4211 5 107
8314
2.5 108
3947 2.5 107
10,713
1 108
4779 1 107
9799
5 109
4016 5 108
8248
2.5 109
3174 2.5 108
8104
1 109
2979 1 108
7921
5 1010
1608 5 109
5172
2.5 1010
842 2.5 109
3090
1 1010
414 1 109
922
5 1011
220 5 1010
516
2.5 1011
212 2.5 1010
515
1 1011
183 1 1010
565
5 1012
188 5 1011
478
2.5 1012
179 2.5 1011
477
1 1012
192 1 1011
494
5 1013
176 5 1012
482
2.5 1013
190 2.5 1012
481
RLU, relative light unit
36 Tingting Xu et al.](https://image.slidesharecdn.com/23858053-250523044023-b9fb45dd/85/Bioluminescent-Imaging-Methods-And-Protocols-1st-Steven-Ripp-49-320.jpg)
Bioluminescent Imaging Methods And Protocols 1st Steven Ripp
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- 6. M E T H O D S I N M O L E C U L A R B I O L O G Y Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK For further volumes: http://www.springer.com/series/7651
- 7. For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by- step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.
- 8. Bioluminescent Imaging Methods and Protocols Edited by Steven Ripp The Center for Environmental Biotechnology, The University of Tennessee, Knoxville, TN, USA 490 BioTech Inc., Knoxville, TN, USA
- 9. Editor Steven Ripp The Center for Environmental Biotechnology The University of Tennessee Knoxville, TN, USA 490 BioTech Inc. Knoxville, TN, USA ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-4939-9939-2 ISBN 978-1-4939-9940-8 (eBook) https://doi.org/10.1007/978-1-4939-9940-8 © Springer Science+Business Media, LLC, part of Springer Nature 2020 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.
- 10. Preface Bioluminescent imaging uses the power of light to visualize, monitor, measure, and track biological processes as they occur in vitro in the laboratory test tube, in vivo in the live animal, ex vivo in animal tissues, or in planta in intact plants. This imaging modality has seen widespread application to address basic research questions in cellular and molecular biology and has evolved as a reporter gene toolset now commonly used in biomedical fields (pathogenesis, oncology, immunology, neurology) and for environmental biomonitoring. The chapters in this volume provide detailed protocols and methodologies required to perform bioluminescent imaging at all of its multifaceted stages to enable the reader to integrate this technology into their laboratory-based imaging experiments. Knoxville, TN, USA Steven Ripp v
- 11. Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix PART I IN VITRO/EX VIVO IMAGING APPLICATIONS 1 High-Throughput Bioluminescence Imaging and Reporter Gene Assay with 3D Spheroids from Human Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Maria Maddalena Calabretta, Laura Montali, Antonia Lopreside, Elisa Michelini, and Aldo Roda 2 Bioluminescence Resonance Energy Transfer (BRET) Coupled Near-Infrared Imaging of Apoptotic Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Setsuko Tsuboi and Takashi Jin 3 High-Throughput Analysis of Endocrine-Disrupting Compounds Using BLYES and BLYAS Bioluminescent Yeast Bioassays . . . . . . . . . . . . . . . . . . . 29 Tingting Xu, Anna Young, Jasleen Narula, Gary Sayler, and Steven Ripp 4 Bioluminescent Imaging of Single Bacterial Cells Using an Enhanced ilux Operon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Carola Gregor 5 Bioluminescent Imaging and Tracking of Bacterial Transport in Soils . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53 Jie Zhuang, Weipeng Liu, Liqiong Yang, Jia Kang, and Xiaoming Zhang PART II IN VIVO IMAGING OF MICROBIAL PATHOGENESIS 6 In Vivo Bioluminescent Imaging of Yersinia ruckeri Pathogenesis in Fish . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69 José A. Guijarro and Jessica Méndez 7 In Vivo Bioluminescent Monitoring of Parasites in BALB/c Mouse Models of Cutaneous Leishmaniasis Drug Discovery . . . . . . . . . . . . . . . . . 81 Diana Caridha, Susan Leed, and Alicia Cawlfield 8 Multiplex Imaging of Polymicrobial Communities—Murine Models to Study Oral Microbiome Interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107 Jens Kreth, Yasser M. Abdelrahman, and Justin Merritt 9 Bioluminescence Imaging to Study Mature Biofilm Formation by Candida spp. and Antifungal Activity In Vitro and In Vivo . . . . . . . . . . . . . . . . . . 127 Katrien Van Dyck, Patrick Van Dijck, and Greetje Vande Velde vii
- 12. PART III IN VIVO SMALL ANIMAL/ORGANISM IMAGING APPLICATIONS 10 Bioluminescence Imaging of Neuroinflammation in a Mouse Model of Parkinson’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147 Maria Eugenia Bernis and Gültekin Tamgüney 11 Continual Conscious Bioluminescent Imaging in Freely Moving Mice . . . . . . . . 161 Juan Antinao Diaz, Amy Geard, Lorna M. FitzPatrick, Juliette M. K. M. Delhove, Suzanne M. K. Buckley, Simon N. Waddington, Tristan R. McKay, and Rajvinder Karda 12 In Vivo Bioluminescent Imaging of Marburg Virus in a Rodent Model. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177 Shan Lei, Weijin Huang, Youchun Wang, and Qiang Liu 13 Continuous and Real-Time In Vivo Autobioluminescent Imaging in a Mouse Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 Derek Yip, Andrew Kirkpatrick, Tingting Xu, Tom Masi, Stacy Stephenson, Steven Ripp, and Dan Close 14 In Vivo Tracking of Tumor-Derived Bioluminescent Extracellular Vesicles in Mice. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 Prakash Gangadaran and Byeong-Cheol Ahn 15 Bioluminescence Imaging in the Chick Chorioallantoic Membrane Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Benedict Jefferies, Zhichao Tong, and Roman Nawroth 16 Application of a cybLuc Aminoluciferin for Deep Tissue Bioluminescence Imaging in Rodent Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 Xiang Li and Minyong Li PART IV IN PLANTA IMAGING APPLICATIONS 17 Application of Single-Cell Bioluminescent Imaging to Monitor Circadian Rhythms of Individual Plant Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231 Tomoaki Muranaka and Tokitaka Oyama Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243 viii Contents
- 13. Contributors YASSER M. ABDELRAHMAN • Department of Restorative Dentistry, School of Dentistry, Oregon Health and Science University, Portland, OR, USA; Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt BYEONG-CHEOL AHN • Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu, Republic of Korea; Department of Nuclear Medicine, Kyungpook National University Hospital, Daegu, Republic of Korea MARIA EUGENIA BERNIS • German Center for Neurodegenerative Diseases (DZNE), Bonn, Germany SUZANNE M. K. BUCKLEY • Gene Transfer Technology Group, Institute for Women’s Health, University College London, London, UK MARIA MADDALENA CALABRETTA • Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna, Italy DIANA CARIDHA • Walter Reed Army Institute of Research, Silver Spring, MD, USA ALICIA CAWLFIELD • Dwight D. Eisenhower Army Medical Center, Fort Gordon, GA, USA DAN CLOSE • 490 BioTech Inc., Knoxville, TN, USA JULIETTE M. K. M. DELHOVE • Robinson Research Institute, University of Adelaide, Adelaide, SA, Australia JUAN ANTINAO DIAZ • Gene Transfer Technology Group, Institute for Women’s Health, University College London, London, UK LORNA M. FITZPATRICK • Centre for Biomedicine, Manchester Metropolitan University, Manchester, UK PRAKASH GANGADARAN • Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu, Republic of Korea; Department of Nuclear Medicine, Kyungpook National University Hospital, Daegu, Republic of Korea AMY GEARD • UCL School of Pharmacy, University College London, London, UK; Wits/ SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa CAROLA GREGOR • Department of NanoBiophotonics, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany JOSÉ A. GUIJARRO • Departamento de Biologı́a Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, Oviedo, Asturias, Spain WEIJIN HUANG • Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, National Institutes for Food and Drug Control, Beijing, China BENEDICT JEFFERIES • Department of Urology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany TAKASHI JIN • Riken Center for Biosystems Dynamics Research, Riken, Osaka, Japan JIA KANG • Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China; The University of Chinese Academy of Sciences, Beijing, China RAJVINDER KARDA • Gene Transfer Technology Group, Institute for Women’s Health, University College London, London, UK ANDREW KIRKPATRICK • 490 BioTech Inc., Knoxville, TN, USA ix
- 14. JENS KRETH • Department of Restorative Dentistry, School of Dentistry, Oregon Health and Science University, Portland, OR, USA SUSAN LEED • Walter Reed Army Institute of Research, Silver Spring, MD, USA SHAN LEI • Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, National Institutes for Food and Drug Control, Beijing, China MINYONG LI • Department of Medicinal Chemistry, Key Laboratory of Chemical Biology of Natural Products (MOE), School of Pharmacy, Shandong University, Jinan, Shandong, China XIANG LI • Department of Medicinal Chemistry, Key Laboratory of Chemical Biology of Natural Products (MOE), School of Pharmacy, Shandong University, Jinan, Shandong, China QIANG LIU • Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, National Institutes for Food and Drug Control, Beijing, China WEIPENG LIU • Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China; The University of Chinese Academy of Sciences, Beijing, China ANTONIA LOPRESIDE • Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna, Italy TOM MASI • Department of Surgery, Graduate School of Medicine, The University of Tennessee Medical Center, Knoxville, TN, USA TRISTAN R. MCKAY • Centre for Biomedicine, Manchester Metropolitan University, Manchester, UK JESSICA MÉNDEZ • Departamento de Biologı́a Funcional, Facultad de Medicina, IUBA, Universidad de Oviedo, Oviedo, Asturias, Spain JUSTIN MERRITT • Department of Restorative Dentistry, School of Dentistry, Oregon Health and Science University, Portland, OR, USA; Department of Molecular Microbiology and Immunology, Oregon Health and Science University, Portland, OR, USA ELISA MICHELINI • Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna, Italy; INBB, Istituto Nazionale di Biostrutture e Biosistemi, Rome, Italy; Health Sciences and Technologies-Interdepartmental Center for Industrial Research (HST-ICIR), University of Bologna, Bologna, Italy LAURA MONTALI • Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna, Italy TOMOAKI MURANAKA • Center for Ecological Research, Kyoto University, Otsu, Shiga, Japan JASLEEN NARULA • The Center for Environmental Biotechnology, The University of Tennessee, Knoxville, TN, USA ROMAN NAWROTH • Department of Urology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany TOKITAKA OYAMA • Department of Botany, Graduate School of Science, Kyoto University, Kyoto, Japan STEVEN RIPP • The Center for Environmental Biotechnology, The University of Tennessee, Knoxville, TN, USA; 490 BioTech Inc., Knoxville, TN, USA ALDO RODA • Department of Chemistry “Giacomo Ciamician”, University of Bologna, Bologna, Italy; INBB, Istituto Nazionale di Biostrutture e Biosistemi, Rome, Italy GARY SAYLER • The Center for Environmental Biotechnology, The University of Tennessee, Knoxville, TN, USA; 490 BioTech Inc., Knoxville, TN, USA STACY STEPHENSON • Department of Surgery, Graduate School of Medicine, The University of Tennessee Medical Center, Knoxville, TN, USA x Contributors
- 15. GÜLTEKIN TAMGÜNEY • Institute of Complex Systems — Structural Biochemistry (ICS-6), Forschungszentrum Jülich GmbH, Jülich, Germany; Institut für Physikalische Biologie, Heinrich-Heine-Universit€ at Düsseldorf, Düsseldorf, Germany ZHICHAO TONG • Department of Urology, Klinikum rechts der Isar, Technical University of Munich, Munich, Germany SETSUKO TSUBOI • Riken Center for Biosystems Dynamics Research, Riken, Osaka, Japan PATRICK VAN DIJCK • VIB-KU Leuven Center for Microbiology, Leuven, Belgium; Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven, Belgium KATRIEN VAN DYCK • VIB-KU Leuven Center for Microbiology, Leuven, Belgium; Laboratory of Molecular Cell Biology, Institute of Botany and Microbiology, KU Leuven, Leuven, Belgium GREETJE VANDE VELDE • Biomedical MRI/MoSAIC, Department of Imaging and Pathology, KU Leuven, Leuven, Belgium SIMON N. WADDINGTON • Gene Transfer Technology Group, Institute for Women’s Health, University College London, London, UK; Wits/SAMRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa YOUCHUN WANG • Division of HIV/AIDS and Sex-Transmitted Virus Vaccines, National Institutes for Food and Drug Control, Beijing, China TINGTING XU • The Center for Environmental Biotechnology, The University of Tennessee, Knoxville, TN, USA LIQIONG YANG • Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China; The University of Chinese Academy of Sciences, Beijing, China DEREK YIP • 490 BioTech Inc., Knoxville, TN, USA ANNA YOUNG • The Center for Environmental Biotechnology, The University of Tennessee, Knoxville, TN, USA XIAOMING ZHANG • College of Desert Control Science, Inner Mongolia Agricultural University, Hohhot, China JIE ZHUANG • Department of Biosystems Engineering and Soil Science, Center for Environmental Biotechnology, The University of Tennessee, Knoxville, TN, USA; Key Laboratory of Pollution Ecology and Environmental Engineering, Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang, China Contributors xi
- 16. Part I In Vitro/Ex Vivo Imaging Applications
- 17. Chapter 1 High-Throughput Bioluminescence Imaging and Reporter Gene Assay with 3D Spheroids from Human Cell Lines Maria Maddalena Calabretta, Laura Montali, Antonia Lopreside, Elisa Michelini, and Aldo Roda Abstract 3D cell culture models represent an attractive approach to decode intracellular and intercellular signaling, providing biologically relevant information and predictive data. Bioluminescent reporter gene assays and bioluminescence imaging in 3D cell models are very promising bioanalytical tools for several applications. Here we report a very straightforward method for bioluminescence imaging and bioluminescent reporter gene assays in 3D cell-culture models. Both the assays can be easily implemented in laboratories equipped with basic cell culture facilities and instrumentation for bioluminescence detection, that is, low-light detectors connected to inverted microscopes and luminometers, without the need for additional equipment. Key words Bioluminescence, Spheroids, Imaging, Reporter gene, 3D cell models, Luciferase 1 Introduction Bioluminescent (BL) cell-based assays based on two-dimensional (2D) cell cultures represent a well-established bioanalytical tool for interrogating natural biological phenomena, including gene expression, promoter regulation, and for monitoring molecular pathways, thus providing highly valuable information. Major appli- cations of these assays are in drug discovery but also span from regenerative medicine to environmental monitoring and food con- trol, as well as forensic applications [1–3]. Several cell-based assays have been developed based on bioluminescent reporters such as luciferases. Reporter gene technology relies on the splicing of transcriptional control elements to a variety of reporter proteins to monitor cellular events associated with gene expression and intracellular signaling cascades. The main advantages of these assays are wide dynamic range, high sensitivity, and suitability to high throughput-screening. Moreover, cell-based assays relying on luci- ferases as reporter protein are generally very simple to implement Steven Ripp (ed.), Bioluminescent Imaging: Methods and Protocols, Methods in Molecular Biology, vol. 2081, https://doi.org/10.1007/978-1-4939-9940-8_1, © Springer Science+Business Media, LLC, part of Springer Nature 2020 3
- 18. and do not require expensive equipment or skilled personnel. More recently, thanks to the availability of luciferases emitting at different colors or requiring different substrates, multiplexed assays have also been developed to monitor two or more analytes or to include viability controls to correct the analytical signal according to cell viability [4, 5]. The recent technical advancements have dramati- cally reshaped the way cell-based assays are developed, expanding their applicability outside laboratories for on-site analyses. Several whole-cell biosensors have been reported in the past years, relying on the use of portable light detectors (such as charge-coupled devices and complementary metal oxide semiconductors) or simply integrated into 3D-printed smartphone-based devices [6–8]. These approaches are surely promising; however, 2D cell cultures do not always resemble the morphology and the functionality of the natu- ral 3D environment. As a result, 2D cell culture tests can provide misleading data, reducing the reliability and predictivity of the results. Instead, 3D cell models are characterized by complex cell- to-cell communication and formation of extracellular matrix (ECM), thus restoring complex tissue architecture [9]. 3D cell culture models represent an attractive approach to decode intra- and intercellular signaling, providing biologically relevant information and predictive data [10]. It is known that cells in 3D culture environments differ in gene, protein, and cell receptor expression from 2D-monolayer cell cultures and they provide an excellent model as surrogate in vivo systems. 3D struc- ture spheroids exhibit enhanced cell viability, stable morphology and polarization, increasing proliferative activity and physiological metabolic function, which are markedly improved when compared to 2D cell monolayers [11, 12]. Besides the European Union, the USA and many other countries are strongly encouraging the implementation of in vitro assays and other approaches to replace animal testing, in accordance with the “three Rs principle” of Russell and Burch [13]. The possibility of exploiting bioluminescence detection for monitoring 3D cell models has been previously demonstrated by us and others [14–17]. We have previously reported a high-throughput BL 3D cell- based assay in micropatterned 96-well plate format using the tran- scriptional regulation of nuclear factor K beta response element in human embryonic kidney (HEK293) cells. Here we report a very straightforward method for bioluminescence imaging and biolumi- nescent reporter gene assays in 3D cell-culture models. Both the assays can be easily implemented in laboratories equipped with basic cell culture facilities and instrumentation for bioluminescence detection, that is, luminometer and low-light detectors connected to inverted microscopes, without the need for additional equipment. 4 Maria Maddalena Calabretta et al.
- 19. 2 Materials 2.1 3D Cell Culture and Transfection 1. Human embryonic kidney HEK293 cell line (ATCC® CRL-1573) or other cell lines that can be transfected readily. 2. Growth Medium: Dulbecco’s modified Eagle’s medium (DMEM) high glucose. Supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 μg/mL penicillin and 50 μg/mL streptomycin (Carlo Erba reagents, Cornaredo, MI, Italy). Filter the growth medium using a filter cup 0.22 μm. Store at 4 C (see Note 1). 3. Trypsin–EDTA solution 1 (Carlo Erba reagents, Cornaredo, MI, Italy). Store at 4 C. 4. T25 flask culture and serological pipettes. 5. 96-well microspace round bottom cell culture plates (Elpla- sia™, Kuraray, Japan). 6. FuGENE HD transfection reagent (Promega, Madison, WI). 7. Plasmids: pGL4.32[PLG2] obtained by replacing Luc2P from vector pGL4.32[luc2P/NF-kB-RE/Hygro] (Promega, Madi- son, WI) with PLG2 luciferase gene [18]; plasmid for constitu- tive expression of the luciferase (e.g., pCMV_PLG2) to characterize luciferase emission in 3D cell cultures. 2.2 Reagents 1. 0.1 M phosphate buffered saline (PBS): Add 8 g of NaCl, 200 mg of KCl, 1.44 g of Na2HPO4, 240 mg of KH2PO4 into 1 L of double-distilled water. Adjust solution to desired pH (typically pH 7.4). Sterilize the solution in autoclave and prepare 50 mL aliquots. Store at room temperature. 2. 1.0 mM D-luciferin solution: Prepare Buffer n.1 (0.1 M citric acid solution) by dissolving 21.0 g of citric acid monohydrate, C6H8O7·H2O into 1 L of distilled water; prepare Buffer n.2 (0.1 M trisodium citrate solution) by dissolving 29.41 g of trisodium citrate dihydrate, C6H5O7Na3·2H2O, into 1 L of distilled water. Mix 35 mL of Buffer n.1 and 65 mL Buffer n.2 for 30 min to obtain citric acid-sodium citrate buffer solution at pH 5.0. Dissolve 28.3 mg of D-luciferin into 100 mL of citric acid–sodium citrate buffer solution at pH 5.0. Prepare 8–10 mL aliquots and store at 20 C (see Note 2). 3. Tumor Necrosis Factor Alpha (TNFα) stock solution: Centri- fuge the vial before opening. Reconstitute the TNFα with 1 mL PBS sterile solution 0.1 M pH 7.4 containing 0.1% endotoxin- free recombinant human serum albumin in order to obtain a stock solution 10.0 μg/mL. Prepare 100 μL aliquots and store at 20 C (see Note 3). Prepare on the day of the assay serial dilutions of TNFα (concentration range 0.1–50.0 ng/mL) using sterile deionized water. Bioluminescence Imaging of 3D Spheroids 5
- 20. 2.3 Equipment and Software 1. For cell cultures: laminar flow cabinet and 5% CO2 incubator maintained at 37 C. 2. For bioluminescence imaging: inverted microscope (Olympus CK40) connected to an electron multiplying charge-coupled device (EMCCD) camera (ImagEM-X2, Hamamatsu). 10 (Olympus A10PL) and 4 (SPlan4SL, 0.16 N.A.) objectives are used for bright-field and BL imaging. 3. Light-tight box to protect the imaging system from light (see Note 4). 4. HCImage software (v 4.1.2.0, Hamamatsu Corporation) for image analysis. 5. For luminometric measurements: Varioskan Flash Lumin- ometer (Thermo Fisher Scientific, Walthman, MA) and Thermo Scientific SkanIT Software for Varioskan Flash (ver- sion 2.4.3). 6. GraphPad Prism software v. 5.02 (GraphPad Software Inc.). 7. ImageJ version 1.51d software for spheroid analysis (https:/ / imagej.nih.gov/ij/). 3 Methods Carry out all procedures at room temperature (25 C) under the laminar flow hood (unless indicated otherwise). 3.1 Spheroids Formation 1. Plate cells in 25 cm2 flasks for cell culture in complete growth medium with serum. Culture cells overnight under standard conditions in preparation for spheroid production the following day. 2. Remove and discard the medium when the cells are approxi- mately 80% confluent (80% of surface of 25 cm2 flask covered by cell monolayer). 3. Gently rinse the cells with 5 mL of PBS 0.1 M pH 7.4 for removing all traces of serum that contains trypsin inhibitor. 4. Add 2.0 mL of prewarmed trypsin–EDTA solution 1 to the side wall of the flask. Observe the cells under inverted micro- scope. Detached cells appear rounded and float in the medium. If less than 80–90% of cells are detached incubate again the flask for an additional 2 min and observe the cells every 30 s. Then add 5 mL of complete fresh growth medium to stop the trypsin action (see Note 5). 5. Transfer the cell suspension to the tube and centrifuge the cells at 1600 g for 8–10 min. 6 Maria Maddalena Calabretta et al.
- 21. 6. Remove the supernatant and add 5 mL of prewarmed complete growth medium, resuspend cells by gently pipetting. 7. Count the cells and plate them in a 96-well microspace round bottom Elplasia cell culture plate at a concentration of 2 104 cells per well in a total volume of 200 μL of medium (see Note 6). 8. Place the plate in incubator at 37 C with 5% CO2 for 48–72 h for spheroid formation (see Note 7). 3.2 Spheroid Analysis 1. Analyze bright-field images of HEK293 spheroids at desired time points (e.g., every 24 h) using ImageJ version 1.51d software to calculate the projected area (A) and perimeter (P) of each spheroid according to [19]. Calculate a sphericity factor (ϕ) as follows: ϕ ¼ π ffiffiffiffiffi 4A π q P 2. Determine the projected area and perimeter for each spheroid using ImageJ and export data to MS EXCEL to calculate the sphericity factor of about 30 spheroids to obtain an average value standard deviation (see Note 8). 3. Calculate the average diameter of 30 spheroids, generally after 72 h of incubation (cell density of 4 104 cells) HEK293 spheroids with an average diameter of 210 25 μm can be obtained (see Note 9). 3.3 Spheroid Transfection Transfect 3-day-old HEK293 spheroids with 0.10 μg of plasmid per well of a 96-well Elplasia plate with Fugene® HD reagent with a ratio of 3:1 (see Note 10). Add a total volume of transfection mix (plasmid DNA and FuGENE® HD) of 2–10 μL per well. For transfecting 10 wells prepare the following mixture. 1. Mix by inverting briefly the Fugene HD reagent (see Note 11). 2. Add sterile deionized water to a sterile polystyrene Eppendorf to reach 62 μL final volume. 3. Add 1.1 μg plasmid in sterile H2O and mix immediately by pipetting. 4. Add 3.3 μL Fugene HD reagent (ratio 3:1) and mix briefly by vortexing. 5. Incubate the Fugene HD reagent/DNA mixture for 5–10 min at room temperature. 6. Add 5 μL of this mixture per well and mix by gently moving the plate (see Note 12). 7. Place the plate in incubator at 37 C, 5% CO2 for 48 h. Bioluminescence Imaging of 3D Spheroids 7
- 22. 3.4 Characterization of 3D HEK293 Expressing PLG2 Luciferase Before performing the bioluminescent reporter gene assay and imaging sessions, characterize luciferase emission in spheroids in terms of emission wavelength and kinetics. These preliminary mea- surements are crucial to identify the optimal temporal window for BL measurements and for characterizing emission behavior in living cells. Transfect 1-day-old HEK293 spheroids with a plasmid for constitutive expression (plasmid pCMV_PLG2), 0.10 μg of per well with Fugene HD transfect reagent using the procedure previ- ously described and incubate for 24 h at 37 C, 5% CO2. For luminometric measurements with Thermo Scientific SkanIT Soft- ware follow the procedure: 1. Create a new session file. 2. Select “Plate layout” and define wells to be measured. 3. Select “Area” and define the area for acquire BL signals in the 96-well plate. 4. Add “Kinetic Loop” for executing the steps multiple times before continuing with the protocol. Select number of readings of in the range 300–600. 5. In the “Dispense” step choose the dispenser, the volume of the substrate (100 μL) and medium dispensing speed. Use the “Dispense at reading” parameter for starting dispensing after 3 readings. While in the luminometric measurements. 6. Add “Luminometric measurement” for kinetic emission. Set the measurement time (range 200–1000 ms). 7. Add “Luminometric scanning” for emission spectrum. Select scanning wavelength start: 450 nm, end 700 nm; select step size 2 nm; dynamic range: autorange; measurement time: 200–1000 ms (see Note 13). 8. Add 100 μL of D-luciferin solution to each well with automatic injection and acquire kinetic measurements for 5–10 min inte- grating BL signals for 200 ms. 9. Export data and analyze results with GraphPad Prism software. Figure 1 shows a typical emission spectrum obtained with luciferase PLG2, characterized by lack of emission color change at low pH (6.5) and high thermostability compared with wild-type Photinus pyralis luciferase (24 h versus 20 min at 37 C) (see Note 14). 3.5 Bioluminescence Imaging of Spheroids For performing bioluminescence imaging of HEK293 spheroids, transfect 3-day-old HEK293 spheroids with 0.10 μg of plasmid encoding a luciferase under regulation of constitutive promoter (e.g., pCMV_PLG2). Imaging session is performed after 48 h posttransfection with HEK293 cells, however, according to differ- ent cell types and transfection protocols the optimal window for BL measurements may vary from 24 to 72 h posttransfection. 8 Maria Maddalena Calabretta et al.
- 23. 1. Acquire bright-field images of the spheroids in the imaging field before acquiring bioluminescence imaging to localize the luminescence signals on the single spheroids (Fig. 2a). 2. Turn on the imaging system and the software on the computer (HCI images software). Wait for 30 min before image acquisi- tion (see Note 15). 3. Place the plate on the microscope stage. 4. Set the EM gain at 4 and the exposure time at 30 ms. 5. Adjust the focus under the bright-field and capture the live image of spheroids using 10 and 4 objectives. 6. Add gently 100 μL of D-luciferin in citrate buffer pH 5.0, wait 30 s and acquire the BL imaging setting the EM gain level at 500 and the exposure time at 30 s (see Note 16). 7. Overlay images with HCI Images software (v 4.1.2.0) (see Note 17). Fig. 1 (a) Normalized emission spectrum and (b) emission kinetic of PLG2 luciferase expressed in HEK293 spheroids obtained in nonlysing condition using D-luciferin buffer citrate substrate. BL signals are acquired with Varioskan Flash luminometer Fig. 2 Bright-field (a), bioluminescence (b), and pseudocolor overlay (c) images of HEK293 cells grown in 3D microplate format and expressing PLG2 luciferase under constitutive promoter regulation Bioluminescence Imaging of 3D Spheroids 9
- 24. 8. Use ImageJ version 1.51d software for calculating area (A) and the perimeter (P) of each spheroid in bright-field. For obtain- ing corrected BL emissions, quantify BL images designing manually a region of interest (ROI) around each spheroid and calculate BL intensities with respect to the projected area. Figure 2b shows an example of BL imaging with bright-field, bioluminescence, and pseudocolor overlay images of HEK293 cells grown in 3D and expressing PLG2 luciferase. 3.6 Bioluminescent Imaging and Luminometric Assay for Inflammatory Pathway Activation As proof of concept of 3D bioluminescence imaging of HEK293 spheroids, an inflammatory reporter gene assay is described. Trans- fect 3-day-old HEK293 spheroids with 0.10 μg of plasmid pNF-kB_PLG2 encoding PLG2 luciferase under the control of the transcriptional regulation of the nuclear factor k beta (NF-kB) response element. After 48 h posttransfection, HEK293 spheroids are incubated with TNFα solutions (concentration range 0.1–50 ng/mL) for 4 h at 37 C, 5% CO2 (Fig. 3). Cell viability is monitored by transfecting separate wells with plasmid pCMV_PLG2 or by cotransfecting with a luciferase emitting at a different wavelength if filters for spectral resolution are available. The detailed protocol is here described: 1. Change the medium adding fresh growth medium until the well is completely filled. 2. Remove gently 240 μL of medium in order to obtain 100 μL of volume per well. 3. Add 50 μL of TNFα solutions in culture medium in order to obtain final concentration range from 0.1 to 20 ng/mL. Add 50 μL of culture medium in the control wells. Perform all experi- ments at least in triplicate and repeat them at least three times. 4. Incubate the plate for 4 h at 37 C, 5% CO2. Fig. 3 Schematic representation of bioluminescence-based imaging assay for inflammatory pathway activa- tion obtained with HEK293 spheroids transfected with pNF-kB_PLG2 vector 10 Maria Maddalena Calabretta et al.
- 25. For the luminometric assay: 1. Acquire the BL emission with Varioskan Flash by creating a new session with Thermo Scientific SkanIT Software as previ- ously described in the Subheading 3.4 setting the parameters for luminometric measurements with automatic injection of 100 μL of D-luciferin. 2. Analyze data with GraphPad Prism software and calculate the fold induction as the ratio between the average relative light units of induced cells and average relative light units of control cells and analyze data with GraphPad Prism software. For the imaging assay: 1. Perform imaging of 3D cell cultures with the ImagEMX2 EMCCD camera using 4 (SPlan4SL) objectives with an inte- gration time of 30 s, at a gain level set to 500, after the addition of 100 μL of 1 mM D-luciferin substrate per well (see Note 18). 2. Manually design the region of interest around each spheroid and calculate BL intensity with respect to the projected area (corrected BL emission). 3. Calculate the mean of corrected BL emissions obtained from 20 spheroids to construct the dose–response curve generated using GraphPad Prism software (Fig. 4). Fig. 4 Dose–response curve and BL images obtained with HEK293 spheroids transfected with 0.10 μg of reporter plasmid pNF-kB_PLG2 and incubated with different concentrations of TNFα for 4 h at 37 C. BL signals are obtained after the addition of nonlysing D-luciferin buffer citrate substrate Bioluminescence Imaging of 3D Spheroids 11
- 26. 4 Notes 1. Warm cell culture media to 37 C prior to use. 2. D-luciferin must be protected from light. Cover the aliquots with aluminum foil before storage at 20 C. 3. Lyophilized TNF remains active for 1 year at 20 C. Upon reconstitution, it can be stored at 2–8 C for short term only, or at 20 C to 80 C in aliquots for long term storage. Avoid repeated freeze–thaw cycles. 4. A customized light-tight box can be fabricated instead of commercial ones. 5. To avoid clumping do not agitate the cells by hitting or shaking the flask while waiting for the cells to detach. 6. Before seeding add 100 μL of complete culture medium to each well, then add 200 μL of cell suspension. 7. Since spheroid formation may vary depending on cell type, check the cells every 24 h. 8. Average values of sphericity factor obtained with Elplasia plates are generally close to 1, meaning that the aggregates are of nearly spherical shape. 9. Select the incubation time according to desired spheroid size which must be in accordance to the goal of the experiment. In order to obtain spheroid without necrosis in the core and to maintain the functionality of the cells within the aggregate an average diameter of 150–200 μm is suggested. Besides, oxygen availability at the core of spheroids represents an issue for luciferase-based assays. 10. Transfection efficiency may vary depending on cell type and transfection reagent. Change transfection reagent–DNA ratio to improve transfection efficiency. Transfection efficiency can be estimated by cotransfecting with a plasmid expressing a green fluorescent protein and measuring the percentage of fluorescent cells. 11. Before use, allow the vial of the transfection reagent to reach room temperature. 12. Before transfection, add fresh growth medium to completely fill the wells, then remove 245 μL of medium in order to obtain a final volume of 100 μL after transfection. 13. The “Measurement time” defines the light collection time used in the measurement. For luminometric intensities a default measurement time of 1000 ms is suggested; however, when luciferases with high specific activity are used lower measure- ment times can be selected (e.g., 200 ms). 12 Maria Maddalena Calabretta et al.
- 27. 14. When bioluminescence measurements are performed within living cells it is of crucial importance to record emission spectra in order to assess eventual changes in luciferase emissions due to low pH or high temperature. In particular, when two luci- ferases emitting at different colors are measured in the same well by spectral resolution pH- and temperature-insensitive luciferases must be selected to avoid emission overlapping. 15. Before imaging, let the EM-CCD camera reach a temperature of 65 C. A stable cooling temperature is essential to obtain superior performance and for stable gain settings. 16. Addition of reagents is a critical step and liquid reagents should be gently added to leave spheroids undisturbed. 17. Acquire the bright-field images before and after the addition of D-luciferin substrate to verify the correct focus. 18. The choice of objectives is critical for detecting bioluminescent images since the light collection power is proportional to the square of numerical aperture and indirectly proportional to the magnification value square. References 1. Cali JJ, Niles A, Valley MP, O’Brien MA, Riss TL, Shultz J (2008) Bioluminescent assays for ADMET. Expert Opin Drug Metab Toxicol 4:103–120 2. Michelini E, Cevenini L, Mezzanotte L, Coppa A, Roda A (2010) Cell-based assays: fuelling drug discovery. Anal Bioanal Chem 398:227–238 3. Cevenini L, Calabretta MM, Calabria D, Roda A, Michelini E (2016) Luciferase genes as reporter reactions: how to use them in molecular biology? Adv Biochem Eng Biotech- nol 154:3–17 4. Michelini E, Cevenini L, Mezzanotte L, Ablamsky D, Southworth T, Branchini BR, Roda A (2008) Combining intracellular and secreted bioluminescent reporter proteins for multicolor cell-based assays. Photochem Photobiol Sci 7(2):212–217 5. Tarnow P, Bross S, Wollenberg L, Nakajima Y, Ohmiya Y, Tralau T, Luch A (2017) A novel dual-color luciferase reporter assay for simulta- neous detection of estrogen and aryl hydrocar- bon receptor activation. Chem Res Toxicol 30 (7):1436–1447 6. Cevenini L, Calabretta MM, Tarantino G, Michelini E, Roda A (2016) Smartphone- interfaced 3D printed toxicity biosensor inte- grating bioluminescent “sentinel cells”. Sen- sors Actuators B Chem 225:249–257 7. Li H, Lopes N, Moser S, Sayler G, Ripp S (2012) Silicon photomultiplier (SPM) detec- tion of low-level bioluminescence for the development of deployable whole-cell biosen- sors: possibilities and limitations. Biosens Bioe- lectron 33(1):299–303 8. Cevenini L, Calabretta MM, Lopreside A, Tarantino G, Tassoni A, Ferri M, Roda A, Michelini E (2016) Exploiting NanoLuc lucif- erase for smartphone-based bioluminescence cell biosensor for (anti)-inflammatory activity and toxicity. Anal Bioanal Chem 408 (30):8859–8868 9. Bahrvand H, Hashemi SM, Kazemi Ashtini S, Farrokhi A (2006) Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro. Int J Dev Biol 50:645–652 10. Antoni D, Burckel H, Josset E, Noel G (2015) Three-dimensional cell culture: a breakthrough in vivo. Int J Mol Sci 16(3):5517–5527 11. Benya PD, Shaffer JD (1982) Dedifferentiated chondrocytes reexpress the differentiated colla- gen phenotype when cultured in agarose gels. Cell 30:215–224 12. Nelson CM, Bissell MJ (2005) Modeling dynamic reciprocity: engineering three- dimensional culture models of breast architec- ture, function, and neoplastic transformation. Semin Cancer Biol 15:342–352 Bioluminescence Imaging of 3D Spheroids 13
- 28. 13. Michelini E, Cevenini L, Calabretta MM, Calabria D, Roda A (2014) Exploiting in vitro and in vivo bioluminescence for the implementation of the three Rs principle (replacement, reduction, and refinement) in drug discovery. Anal Bioanal Chem 406 (23):5531–5539 14. Yasunaga M, Fujita Y, Saito R, Oshimura M, Nakajima Y (2017) Continuous long-term cytotoxicity monitoring in 3D spheroids of beetle luciferase-expressing hepatocytes by nondestructive bioluminescence measurement. BMC Biotechnol 17(1):54. https:/ /doi.org/ 10.1186/s12896-017-0374-1 15. Suzuki T, Kanamori T, Inouye S (2017) Quan- titative visualization of synchronized insulin secretion from 3D-cultured cells. Biochem Biophys Res Commun 486(4):886–892 16. Cevenini L, Calabretta MM, Lopreside A, Branchini BR, Southworth TL, Michelini E, Roda A (2017) Bioluminescence imaging of spheroids for high-throughput longitudinal studies on 3D cell culture models. Photochem Photobiol 93(2):531–535 17. Michelini E, Calabretta MM, Cevenini L, Lopreside A, Southworth T, Fontaine DM, Simoni P, Branchini BR, Roda A (2019) Smartphone-based multicolor bioluminescent 3D spheroid biosensors for monitoring inflam- matory activity. Biosens Bioelectron 123:269–277. https:/ /doi.org/10.1016/j. bios.2018.09.012 18. Branchini BR, Southworth TL, Fontaine DM, Kohrt D, Talukder M, Michelini E, Cevenini L, Roda A, Grossel MJ (2015) An enhanced chi- meric firefly luciferase-inspired enzyme for ATP detection and bioluminescence reporter and imaging applications. Anal Biochem 484:148–153 19. Kelm JM, Timmins NE, Brown CJ, Fussenegger M, Nielsen LK (2003) Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83:173–180 14 Maria Maddalena Calabretta et al.
- 29. Chapter 2 Bioluminescence Resonance Energy Transfer (BRET) Coupled Near-Infrared Imaging of Apoptotic Cells Setsuko Tsuboi and Takashi Jin Abstract Detection of apoptotic cells is crucial for understanding the mechanism of diseases and for therapy development. So far, visible-emitting fluorescent probes such as FITC-labeled Annexin V has been widely used for the detection of apoptotic cells. However, such probes cannot be applied to noninvasive imaging in the near-infrared (NIR) region. Compared with visible light, NIR light is highly permeable in turbid biological samples and tissues. In addition, NIR optical imaging has several advantages such as lower autofluorescence and scattering from biological samples, leading to clearer images with high signal to background ratios. Here, we describe the synthesis and application of bioluminescence resonance energy transfer (BRET)-coupled quantum dots (QDs) for the NIR optical imaging of apoptotic cells. Key words Bioluminescence resonance energy transfer (BRET), Renilla luciferase, Near-infrared imaging (NIR imaging), Quantum dots (QDs), Annexin V, Apoptotic cell 1 Introduction Programmed cell death (apoptosis) plays an important role not only in the homeostasis of normal tissues but also in the pathological conditions of diseased tissues [1, 2]. The detection of apoptotic cells is crucial for understanding the mechanism of those diseases and for therapy development. Deregulation in apoptosis induces numerous diseases such as cancer and cardiovascular and neurode- generative diseases [2, 3]. During the early process of apoptosis [4], morphological changes of the cell membrane induce the asymmet- rical distribution of phosphatidylserine (PS) on the outer mono- layer of the membrane [5, 6]. Annexin V is an endogenous protein having binding ability to PS in the presence of Ca2+ ions, and this protein is widely used as a probe for the detection of apoptotic cells [7]. So far, for the detection of apoptotic cells, Annexin V has been modified with fluorescence, luminescence, magnetic resonance contrast, and radioisotopes [7]. Among these probes, fluorescent- dye labeled probes such as FITC- and Cy3/Cy5-Annexin V [8, 9] Steven Ripp (ed.), Bioluminescent Imaging: Methods and Protocols, Methods in Molecular Biology, vol. 2081, https://doi.org/10.1007/978-1-4939-9940-8_2, © Springer Science+Business Media, LLC, part of Springer Nature 2020 15
- 30. are the most popular for the optical detection of apoptotic cells by using fluorescence microscopy or fluorescence activated cell sort- ing. However, most fluorescence-labeled Annexin V-probes emit at the visible region less than 700 nm. Such visible-emitting probes encounter high background signals arising from autofluorescence of intrinsic fluorescent species in biological samples. We have developed a NIR optical imaging technique for the detection of apoptotic cells using bioluminescence resonance energy transfer (BRET) [10–12] coupled annexin V functionalized NIR-QDs [13, 14]. Compared with fluorescence imaging, BRET- based luminescence imaging does not require external excitations, leading to low autofluorescence from biological samples. In this chapter, we describe the synthesis and application of recombinant Annexin V protein functionalized QDs for BRET-coupled NIR optical detection of apoptotic cells (Fig. 1). This probe is designed to emit NIR luminescence due to BRET from Renilla luciferase (RLuc) [15]-coelenterazine (CTZ) to glutathione (GSH) coated NIR emitting CdSeTe/CdS QDs (GSH-QDs) [16, 17]. 2 Materials 2.1 Synthesis of NIR- Emitting QDs 1. Selenium (powder, 99.999%). 2. Tellurium (shot, 1–2 mm, 99.99%). 3. Cadmium 2,4-pentanedionate (ODPA). 4. Tri-octylphosphine oxide (TOPO). 5. Trioctylphosphine (TOP). 6. Tri-butylphosphine (TBP). 7. Hexadecylamine (HDA, 90%). 8. Sulfur (S, crystalline, 99.999%). 9. Inert gas (nitrogen). 10. Fluorescence spectrometer. Fig. 1 Schematic representation for the preparation of a recombinant protein, HisRLuc-Annexin V functiona- lized QDs (Annexin V·RLuc-QDs), where HisRLuc·Annexin V protein directly binds to the surface of GSH-QDs. BRET occurs from CTZ to QD in an Annexin V·RLuc-QD 16 Setsuko Tsuboi and Takashi Jin
- 31. 2.2 Surface Modification of QDs with Glutathione (GSH) 1. Glutathione (GSH, reduced form). 2. Potassium t-butoxide. 3. Gel filtration column. 4. Aqueous solution of 10 mM Na2CO3. 2.3 Protein Synthesis (HisRLuc-Annexin V) 1. Plasmids containing the sequence of Renilla Mullerei Lucifer- ase (RLuc) and Annexin V. 2. Bacterial vector for high-level expression of proteins with 6His tag, such as pRSET plasmid (ThermoFisher). 3. PCR primers, high-fidelity DNA polymerase, PCR buffer, dNTPs, nuclease-free water and thermal cycler. 4. TAE buffer: 40 mM Tris, 20 mM acetic acid, 1 mM EDTA. 5. 1% Agarose gel: 1% agarose (w/v), TAE buffer. 6. Ethidium Bromide solution (0.5 μg/mL). 7. Gel extraction kit. 8. Cloning enzymes such as ligase and restriction enzymes. 9. Competent cells for protein expression, SOC medium. 10. LB plates with antibiotic appropriate for the plasmid. 11. Plasmid extraction kit. 12. Sequencing system. 13. LB medium, antibiotics, L-Rhamnose, isopropyl β-D-1-thioga- lactopyranoside (IPTG). 14. Binding Buffer: 50 mM Tris, 500 mM NaCl, 20 mM imidazole. 15. Elution Buffer: 50 mM Tris, 500 mM NaCl, 500 mM imidazole. 16. Protease inhibitor. 17. Ni Sepharose 6 Fast Flow (GE Healthcare). 18. Gel filtration column. 19. Phosphate-Buffered Saline (PBS). 20. Ultrasonic homogenizer, Ultracentrifuge, Rotator, Spectrophotometer. 21. SDS-PAGE apparatus. 5–20% polyacrylamide gel, Tris-glycine- SDS buffer, Coomassie Brilliant Blue. 2.4 Bioluminescence Spectral Measurements 1. Aqueous solution of HisRLuc-Annexin V (1 mg/mL in PBS). 2. Ethanol solution (1 mg/mL) of coelenterazine (CTZ). 3. Phosphate-Buffered Saline (PBS). 4. Luminescence spectrometer. BRET Coupled Near-Infrared Imaging 17
- 32. 2.5 Protein Conjugation to GSH- QDs 1. Aqueous solution of HisRLuc-Annexin V (1 mg/mL in PBS). 2. Aqueous solution of GSH-QDs (1 μM in 10 mM Na2CO3, O.D. ¼ ca. 3 at 488 nm). 3. PBS. 4. Gel filtration column. 2.6 BRET Spectra of Annexin V·RLuc-QDs 1. Aqueous solution of Annexin V·RLuc-QDs (1 μM in PBS). 2. PBS. 3. Ethanol solution (1 mg/mL) of coelenterazine (CTZ). 4. Luminescence spectrometer. 2.7 Cell Preparation and Apoptosis Induction 1. Desired cell line for biologic question of interest. 2. General apparatus and reagents used for cell culture. Medium, culture dish, incubator, microscope, and so on. 3. Actinomycin D (ActD). 2.8 Flow Cytometric Analysis and Cell Imaging 1. 1 Annexin V binding buffer (2.5 mM CaCl2 in medium). 2. FITC-Annexin V. 3. Propidium iodide (PI) solution. 4. DMSO solution of ActD. 5. Flow cytometer. 2.9 BRET-Coupled Luminescence Imaging of Apoptotic Cells 1. Aqueous solution of Annexin V·RLuc-QDs (1 μM in PBS). 2. Ethanol solution (1 mg/mL) of CTZ. 3. Luminescence imaging system. 3 Methods NIR-emitting CdSeTe/CdS (core/shell) QDs are prepared by a high temperature decomposition method [16, 17]. A recombinant Annexin V protein (HisRLuc·Annexin V) was prepared by the transformation of pRSET-RLuc-Annexin V plasmid into E. coli. HisRLuc·Annexin V protein has six histidine tags which directly bind to the surface of GSH capped CdSeTe/CdS QDs (Fig. 1). This is due to the high affinity of histidine to Cd2+ ions at the surface of a Cd-S shell layer [18–20]. The GSH capped CdSeTe/ CdS QDs (GSH-QDs) show a broad absorption spectrum from NIR to visible region (400–800 nm) to be able act as an acceptor for the CTZ emission (around 485 nm). 18 Setsuko Tsuboi and Takashi Jin
- 33. 3.1 QD Synthesis (CdSeTe/CdS) Carry out all procedures for QD synthesis in a fume hood, unless otherwise specified. 3.1.1 Se-Te Stock Solution 1. Dissolve Se (24 mg, 0.3 mmol) and Te (13 mg, 0.1 mmol) in TBP (1 mL) at room temperature (see Note 1). 2. Preserve the Se-Te stock solution under a nitrogen atmosphere. 3.1.2 CdSeTe Core 1. Load a mixture of cadmium 2,4-pentanedionate (150 mg, 0.48 mmol), ODPA (300 mg, 0.90 mmol), TOPO (1 g), HDA (3 g), and TOP (0.5 mL) into a 25 mL three-necked flask and heated to 330 C under a nitrogen atmosphere. 2. At this temperature, 1 mL of a Se-Te stock solution was quickly injected by using a syringe, which causes an immediate change in the solution color from colorless to brown. 3. By monitoring the fluorescence spectra, check the formation of CdSeTe QDs (ca. 790 nm emission) (see Note 2). After the CdSeTe QDs were formed, cool the solution to 60 C, and add chloroform (10 mL) to the solution. 4. Precipitate CdSeTe QDs by the addition of methanol. 5. Separate the QDs by centrifugation (see Note 3). 6. Dissolve the QDs in 2 mL of TBP. 3.1.3 Cd-S Stock Solution 1. Dissolve sulfur (40 mg, 1.25 mmol) in TBP (10 mL) at 100 C. After sulfur is completely dissolved, cool the solution to room temperature. 2. Add cadmium 2,4-pentanedionate (388 mg, 1.25 mmol) to the sulfur solution, and warm the solution at 100 C to dissolve cadmium 2,4-pentanedionate. 3. Preserve the Cd-S stock solution under a nitrogen atmosphere. 3.1.4 CdS Overcoating 1. Load a TBP solution of CdSeTe QDs and HDA (3 g) into a 25 mL three-necked flask and heat to 250 C. 2. Add a Cd-S stock solution (0.25 mL) to the solution of CdSeTe/CdS QDs. 3. Check the formation of QDs (ca. 830 nm emission) by moni- toring the fluorescence spectra. After the QDs are formed, cool the solution to 50 C. 4. Add chloroform (10 mL) to the solution. 5. Precipitate CdSeTe/CdS QDs by addition of methanol. 6. Separate CdSeTe/CdS QDs by centrifugation (see Note 3). 7. Dissolve the CdSeTe/CdS QDs into 20 mL of tetrahydrofuran. BRET Coupled Near-Infrared Imaging 19
- 34. 3.2 Surface Modification of QDs with Glutathione (GSH) 1. Add 1 mL of an aqueous solution (50 mg/mL of GSH) to a tetrahydrofuran solution (2 mL) of CdSeTe/CdS QDs (1 μM, O.D. ¼ ca. 3 at 488 nm) at room temperature under sonication. 2. Separate QD precipitates by centrifugation. 3. Add 2 mL of an aqueous solution of potassium t-butoxide (20 mg/1 mL) to the QD precipitates. 4. Sonicate the solution for 5 min. 5. Filter the solution through a 0.45 μm membrane filter to remove excess GSH and potassium t-butoxide. 6. Dialysis the solution with an aqueous solution of Na2CO3 (10 mM) using a 50,000 MW membrane (see Note 4). 7. Measure the fluorescence spectra and transmission electron microscope image of purified GSH-QDs (Fig. 2). 3.3 Protein Synthesis (HisRLuc·Annexin V) 1. Insert the DNA fragment of RLuc and Annexin V amplified by PCR into a bacterial vector. 2. Transform the constructed HisRLuc·Annexin V plasmid into competent cells for protein expression. 3. Grow precultures overnight in LB medium containing antibi- otic appropriate for the plasmid at 37 C. 4. Dilute overnight precultures 1:100 into LB containing antibi- otic appropriate for the plasmid and grow cultures at 37 C until their density (O.D.600) reach to 0.5–0.6. 5. When the cultures reach an O.D.600 of 0.5–0.6, shift temper- ature to 15–25 C and induce protein expression by adding Fig. 2 (a) Fluorescence spectra of GSH-QDs in water. (b) Transmission electron spectra of GSH-QDs. Scale bar: 20 nm 20 Setsuko Tsuboi and Takashi Jin
- 35. isopropyl β-D-1-thiogalactopyranoside (0.2 mM) and L-Rham- nose (0.1%). 6. Grow the cultures overnight at 15–25 C with shaking at 190 rpm. 7. Collect the cells by centrifugation at 5000 g for 10 min. 8. Resuspend the cell pellet in binding buffer with protease inhib- itor added just prior to use. 9. Sonicate the cell suspension using a 10 s bursts at middle- intensity with a cooling period of 10 s between each burst on ice. 10. Centrifuge the lysate at 20,000 g for 20 min to eliminate cell debris. 11. Purify according to the instructions of Ni Sepharose 6 Fast Flow. 12. Exchange the buffer of eluted fractions with PBS using a gel filtration column. 13. Confirm the purified HisRLuc·Annexin V protein by SDS-PAGE (Fig. 3). A size marker is used to compare the molecular weight of HisRLuc·Annexin V protein. The expected molecular size for HisRLuc·Annexin V protein was 76 kDa. Fig. 3 SDS-PAGE of HisRLuc-Annexin V BRET Coupled Near-Infrared Imaging 21
- 36. 3.4 Bioluminescence Spectra for HisRLuc·Annexin V 1. Add 20 μL of an aqueous solution of HisRLuc·Annexin V (1 mg/mL) to 2 mL of PBS. 2. Add 10 μL of an ethanol solution (1 mg/mL) of CTZ. 3. Measure bioluminescence spectra using a luminescence spec- trometer (Hamamatsu Photonics) with 1 min exposure time (Fig. 4, see Note 5). 3.5 Protein Conjugation to GSH- QDs 1. Add 300 μL of HisRLuc·Annexin V (1 mg/mL in PBS) to 0.4 mL of GSH-QDs (1 μM in 10 mM Na2CO3 solution). 2. Exchange the solution buffer with PBS by using a gel filtration column (PD-10 column, GE Healthcare). 3. Check the purity of HisRLuc·Annexin V conjugated QDs by agarose gel electrophoresis (Fig. 5a). 3.6 BRET Spectra of Annexin V·RLuc-QDs 1. Add 20 μL of an aqueous solution of Annexin V·RLuc-QDs (1 μM) to 2 mL of PBS. 2. Add 10 μL of an ethanol solution (1 mg/mL) of CTZ. 3. Measure BRET spectra using a luminescence spectrometer (Hamamatsu Photonics) with 1 min exposure time (Fig. 5b) (see Note 5). 3.7 Cell Preparation and Apoptosis Induction 1. Culture a cell line in a suitable medium for 24 h. 2. Optimal induction conditions are found by adding aqueous solutions of ActD (0–10 μg/mL) in the target cell line for 24 h and use it as a positive control (see Note 6). Fig. 4 Bioluminescence spectra for HisRLuc-Annexin V 22 Setsuko Tsuboi and Takashi Jin
- 37. 3.8 Flow Cytometric Analysis and Cell Imaging 1. Collect the culture medium in a 15 mL conical tube. 2. Wash the cells with PBS, collect this wash solution and com- bine with the collected culture medium from step 1. 3. Collect cells by trypsinization and mix with the cell suspension collected in steps 1 and 2. 4. Pellet these cells by centrifugation (300 g, 3 min), discard the supernatant, resuspend the cell pellet in 1 Annexin V binding buffer at a concentration of ~106 cells/mL. 5. Transfer 100 μL of the solution to a 1.5 mL sample tube. 6. Add 5 μL of an aqueous solution of FITC-Annexin V and 5 μL an aqueous solution of PI. Incubate for 15 min at room tem- perature in the dark. 7. Add 400 μL of 1 Annexin V binding buffer to each tube and pass through a 40-μm cell strainer mesh. 8. Analyze by flow cytometry as soon as possible (Fig. 6) (see Note 7). 9. Transfer a portion of the cell suspension used for flow cyto- metric analysis to a 35 mm glass bottom dish (D 11134 H, Matsunami) and observe the fluorescence of FITC-Annexin V an FITC filter (Fig. 7). Fig. 5 (a) Agarose gel electrophoresis of GSH-QDs and the mixture of GSH-QDs/ HisRLuc-Annexin V (1:10). (b) BRET spectra of Annexin V·RLuc-QDs in the presence CTZ BRET Coupled Near-Infrared Imaging 23
- 38. 3.9 BRET-Coupled Luminescence Imaging of Apoptotic Cells 1. Add Annexin V·RLuc-QDs (final concentration of 30 nM) to the cell suspension collected similarly to flow cytometry (see Note 8). 2. Incubate for 15 min at room temperature in the dark. 3. Wash the cell suspension with 1 Annexin V binding buffer three times and resuspend the cells with 50 μL of PBS. 4. Immediately before the detection of their NIR luminescence, add CTZ (final concentration of 50 μM) to the cell suspension. Fig. 6 Flow cytometric analysis for HeLa cells treated without and with ActD. Images were taken after incubation with ActD (0, 0.01, 0.1, and 1 μg/mL) for 24 h. Green and red fluorescence shows the emission form FITC-Annexin V and PI, respectively 24 Setsuko Tsuboi and Takashi Jin
- 39. 5. Take BRET-coupled NIR images (at 830 20 nm) by using a luminescence imaging system (Bruker, MS FX PRO) with 10 min exposure time (Fig. 8). 4 Notes 1. Use a freshly prepared solution of TBP. When the Se and Te are not completely dissolved in TBS, sonicate the TBP solution. 2. For this purpose, we use a fluorescence spectrometer nearby a fume food and monitor the fluorescence spectra of QDs. 3. For the separation of QDs, we employ test (glass) tubes for centrifugation. Control the speed of centrifugation so as not to damage the test (glass) tubes. 4. For the exchange of the aqueous solution of QDs with Na2CO3 (10 mM), a gel filtration column (PD-10 column, GE Healthcare) can also be used. 5. To obtain bioluminescence spectra for HisRLuc·Annexin V, the spectra should be measured immediately after the addition of CTZ to the aqueous solution of HisRLuc·Annexin V. 6. In the case of HeLa cells, Actinomycin D (1 μg/mL) is added to induce apoptosis in about 60% of the cells by induction for 24 h. Fig. 7 Cellular imaging of HeLa cells treated without and with ActD. Images were taken after incubation with ActD for 24 h. Green and red fluorescence shows the emission from FITC-Annexin V and PI, respectively BRET Coupled Near-Infrared Imaging 25
- 40. 7. Flow cytometric analysis is performed using the MACSQuant Analyzer (Miltenyi Biotec Inc.). PI is collected through a FL4 filter (ex: 488 nm, em: 655–730 nm band-pass), FITC- Annexin V fluorescence is collected through a FL2 (FITC) filter (ex: 488 nm, em: 525 50 nm). 8. Just before use, mix 1 μM of GSH-QDs and 1 mg/mL of HisRLuc·Annexin V at a volume ratio of 2:1. References 1. Taylor RC, Cullen SP, Martin SJ (2008) Apo- ptosis: controlled demolition at the cellular level. Nat Rev Mol Cell Biol 9:231–241 2. Fuchs Y, Steller H (2015) Live to die another way: modes of programmed cell death and the signals emanating from dying cells. Nat Rev Mol Cell Biol 16:329–344 3. Elmore S (2007) Apoptosis: a review of pro- grammed cell death. Toxicol Pathol 35:495–516 4. Fuchs Y, Steller H (2011) Programmed cell death in animal development and disease. Cell 147:742–758 Fig. 8 BRET coupled NIR imaging of HeLa cells (~106 cells) after incubation with Annexin V·RLuc-QDs. (a) HeLa cells treated without and with ActD (10 μg/mL). (b) HeLa cells treated with 0.01, 0.1, and 1 μg/mL of ActD. Scale bar: 10 mm 26 Setsuko Tsuboi and Takashi Jin
- 41. 5. van Genderen HO, Kenis H, Hofstra L, Narula J, Reutelingsperger CP (2008) Extra- cellular annexin A5: functions of phosphatidylserine-binding and two-dimensional crystallization. Biochim Bio- phys Acta 1783:953–963 6. Lizarbe MA, Barrasa JI, Olmo N, Gavilances F, Turnay J (2013) Annexin-phospholipid inter- actions. Functional implications. Int J Mol Sci 14:2652–2683 7. Nazari M, Minai-Tehrai A, Emamzadeh R (2014) Comparison of different probes based on labeled annexin V for detection of apopto- sis. RSC Adv 4:45128–45135 8. Koopman G, Reutelingsperger C, Kuijten G, Keehnen R, Pals S, Van Oers M (1994) Annexin V for flow cytometric detection of phosphatidylserine expression on B cells under- going apoptosis. Blood 84:1415–1420 9. Petrovsky A, Schellenberger E, Josephson L, Weissleder R, Bogdanov A Jr (2003) Near- infrared fluorescent imaging of tumor apopto- sis. Cancer Res 63:1936–1942 10. Hasegawa M, Tsukasaki Y, Ohyanagi T, Jin T (2013) Bioluminescence resonance energy transfer coupled near-infrared quantum dots using GST-tagged luciferase for in vivo imag- ing. Chem Commun 49:228–230 11. Samanta A, Walper SA, Susumu K, Dwyer CL, Medinz IL (2015) An enzymatically-sensitized sequential and concentric energy transfer relay self-assembled around semiconductor quan- tum dots. Nanoscale 7:7603–7614 12. Yu X, Wen K, Wang Z, Zhang X, Li C, Zhnag S, Shen J (2016) General biolumines- cence resonance energy transfer homogeneous immunoassay for small molecules based on quantum dots. Anal Chem 88:3512–3520 13. Kamkaew A, Sun H, England CG, Cheng L, Liu Z, Cai W (2016) Quantum dot–NanoLuc bioluminescence resonance energy transfer enables tumor imaging and lymph node mapping in vivo. Chem Commun 52:6997–7000 14. Weissleder R (2001) A clearer vision for in vivo imaging. Nat Biotechnol 19:316–317 15. Srikantha T, Klapach A, Lorenz WW, Tsai LK, Laughlin LA, Gorman JA, Soll DR (1996) The sea pansy Renilla reniformis luciferase serves as a sensitive bioluminescent reporter for differ- ential gene expression in Candida albicans. J Bacteriol 178:121–129 16. Jin T, Yoshioka Y, Fujii F, Komai Y, Seki J, Seiyama A (2008) Gd3+ -functionalized near- infrared quantum dots for in vivo dual modal (fluorescence/magnetic resonance) imaging. Chem Commun 44:5764–5766 17. Jin T, Fujii F, Komai Y, Seki J, Seiyama A, Yoshioka Y (2008) Preparation and characteri- zation of highly fluorescent, glutathione- coated near infrared quantum dots for in vivo fluorescence imaging. Int J Mol Sci 9:2044–2061 18. Medintz IL, Uyeda HT, Goldman ER, Mat- toussi H (2005) Quantum dot bioconjugates for imaging, labelling and sensing. Nat Mater 4:435–446 19. Lao UL, Mulchandani A, Chen W (2006) Sim- ple conjugation and purification of quantum dot-antibody complexes using a thermally responsive elastin-protein L scaffold as immu- nofluorescent agents. J Am Chem Soc 128:14756–14757 20. Alam R, Karam LM, Doane TL, Zylstra J, Fon- taine DM, Branchini BR, Maye MM (2014) Near infrared bioluminescence resonance energy transfer from firefly luciferase-quantum dot bionanoconjugates. Nanotechnology 25:495606. (7pp) BRET Coupled Near-Infrared Imaging 27
- 42. Chapter 3 High-Throughput Analysis of Endocrine-Disrupting Compounds Using BLYES and BLYAS Bioluminescent Yeast Bioassays Tingting Xu, Anna Young, Jasleen Narula, Gary Sayler, and Steven Ripp Abstract Bioluminescent yeast assays BLYES and BLYAS are whole-cell bioassays that utilize genetically modified Saccharomyces cerevisiae bioreporters to detect estrogenic and androgenic activities, respectively. The bioreporter strains chromosomally express human estrogen receptor alpha (BLYES) or androgen receptor (BLYAS) and contain a reporter plasmid expressing the complete bacterial luciferase gene cassette (lux- CDABE) under the control of an estrogen- or androgen-responsive promoter. Exposure to endocrine- disrupting compounds activates the receptor which subsequently turns on the expression of the reporter genes, resulting in dose-dependent bioluminescence (i.e., light) emission. These yeast whole-cell bioassays provide rapid, cost-effective, and high-throughput detection of endocrine-disrupting activities in environ- mental samples. This protocol will provide a detailed description of the standard assay procedures as well as a framework for data analysis. Key words Endocrine-disrupting compounds, Emerging contaminants, Bioluminescence, Bacterial luciferase, Bioreporter, High-throughput screening 1 Introduction Endocrine-disrupting compounds (EDCs) are a group of emerging contaminants that are of substantial animal and human health concern due to their potential to interfere with natural endocrine signaling activities. The vertebrate endocrine system utilizes small signaling molecules (hormones) to transcriptionally regulate the expression of a sophisticated gene network throughout different stages of an animal’s life cycle including development, growth and reproduction. EDCs, however, can mimic or repress native hor- mone functions, resulting in an imbalance in the delicate regulation of the endocrine network in humans and wildlife. Mounting scien- tific evidence has demonstrated that exposure to EDCs can lead to reproductive, developmental, and metabolic disorders inclusive of breast, ovarian, testicular, and prostate cancers, early onset of Steven Ripp (ed.), Bioluminescent Imaging: Methods and Protocols, Methods in Molecular Biology, vol. 2081, https://doi.org/10.1007/978-1-4939-9940-8_3, © Springer Science+Business Media, LLC, part of Springer Nature 2020 29
- 43. puberty, reduced sperm counts, and various other adverse health effects [1, 2]. Unfortunately, many compounds that make up everyday consumer products such as plastics, household and per- sonal care products, pharmaceuticals, and pesticides have been found to be EDCs, and their wide use and discharge into the environment through anthropogenic activities and industrial waste has made monitoring of EDCs in the aqueous ecosystem a critical checkpoint to safeguard the health of humans and wildlife. Analytical methods such as gas chromatography/mass spec- trometry are the traditional gold standards for EDC detection. However, despite their supreme sensitivity, analytical methods are expensive, time-consuming, require specialized instruments and personnel, and most importantly, lack the capability to predict EDC bioavailability. As a complement to analytical methods, a variety of whole-cell bioassays have been developed to provide a biological evaluation of the cumulative effects of EDCs in environ- mental samples without identifying individual compounds. Using the lower eukaryotic organism Saccharomyces cerevisiae as a plat- form for EDC screening, two bioluminescent yeast assays BLYES [3] and BLYAS [4] have been developed to detect estrogenic and androgenic activity, respectively. These genetically engineered S. cerevisiae strains chromosomally express a hormone-specific nuclear receptor (i.e., human estrogen receptor alpha in BLYES and human androgen receptor in BLYAS), which upon activation by EDC exposure will turn on the episomal expression of a reporter operon under the control of a hormone-specific promoter. In particular, BLYES and BLYAS express the complete Photorhabdus luminescens luciferase gene cassette (luxCDABE) that allows for autonomous bioluminescent production in a dose-dependent man- ner without the need of exogenous substrate addition or photonic excitation. A constitutively bioluminescent strain BLYR [5] has also been developed and is often used in parallel with BLYES and/or BLYES strains to assess the toxicity of environmental samples. These yeast bioassays can be conveniently performed in test tubes for low-throughput applications or in microtiter plates (i.e., 96-well plates) for simultaneous analysis of multiple samples in a single assay. They provide rapid (usually within a few hours), cost- effective, and high-throughput detection of EDCs and have been successfully applied worldwide for chemical screening and evalua- tion [6–9] and EDC monitoring in surface and drinking water [10, 11] and wastewater [12, 13]. The bioluminescent signal is easily detectable by conventional luminometers for low-throughput applications, or by microplate readers with lumi- nescence capability for high-throughput assays. Here we present the detailed protocol of using the BLYES, BLYAS, and BLYR bioluminescent yeast bioreporters in a 96-well plate format to analyze EDC contaminants in environmental samples. 30 Tingting Xu et al.
- 44. 2 Materials 2.1 Yeast Strains Bioluminescent yeast bioreporter strains used in this protocol include Saccharomyces cerevisiae BLYES for estrogenicity detection, BLYAS for androgenicity detection, and BLYR for toxicity evalua- tion [3, 4, 6]. 2.2 Glassware Glassware must be used for medium preparation and storage and yeast growth. Wrap glassware openings with aluminum foil and sterilize by baking at 500 C for 4 h in a muffle furnace (see Note 1). 2.3 Chemical Standards Serial dilutions of 17β-estradiol (E2) and 5α-dihydrotestosterone (DHT) are used as positive controls and calculation standards for BLYES estrogenicity and BLYAS androgenicity detection, respec- tively. Methanol (HPLC grade) is used as solvent and negative control in BLYES and BLYAS assays. Chemical standards are prepared as below. 1. Dissolve 13.67 mg E2 in 5 mL of methanol in a borosilicate glass vial to make a 1 102 M E2 stock. 2. Serially dilute the 1 102 M E2 stock by tenfold in separate glass EPA vials with methanol to final concentrations of 1 103 M, 1 104 M, 1 105 M, 1 106 M, 1 107 M, 1 108 M, 1 109 M, 1 1010 M, and 1 1011 M. 3. Dilute the 1 106 M E2 stock by twofold with methanol to 5 107 M. 4. Perform 5 tenfold serial dilutions of the 5 107 M E2 solution in separate glass EPA vials with methanol to final concentrations of 5 108 M, 5 109 M, 5 1010 M, 5 1011 M, and 5 1012 M. 5. Dilute the 5 107 M E2 solution by twofold with methanol in a glass vial to 2.5 107 M. 6. Perform 5 tenfold serial dilutions of the 2.5 107 M E2 solution in separate glass EPA vials with methanol to final concentrations of 2.5 108 M, 2.5 109 M, 2.5 1010 M, 2.5 1011 M, and 2.5 1012 M. 7. The final full series of E2 standards will include 18 dilutions, ranging from 1 106 M to 2.5 1012 M. 8. To prepare DHT standards, first dissolve 14.52 mg DHT into 5 mL methanol to generate a 1 102 M stock. 9. From the 1 102 M DHT stock, follow similar dilution patterns in Subheading 2.3 steps 2–6 to generate a full series of DHT standards consisting of 18 dilutions ranging from 1 105 M to 2.5 1011 M (see Note 2). 10. Store E2 and DHT standards at 4 C. Monitoring Emerging Contaminants Through Microbial Bioreporters 31
- 45. 2.4 Medium Preparation All yeast strains should be grown in yeast minimal medium (YMM) without leucine, uracil, or tryptophan for optimal detection sensi- tivity (Table 1). 1. Prepare a 0.8 g/L FeSO4 and a 20 mM CuSO4 solution with HPLC-grade water. Sterilize each by filtration through a 0.2 μm filter membrane into separate sterile, baked glass bot- tles. Store at room temperature. Table 1 Yeast minimal medium (YMM) without leucine, uracil, or tryptophan for the BLYES, BLYAS, and BLYR assay Component Concentration Ammonium sulfate ((NH4)2SO4) 1.7 g/L Copper(II) sulfate (CuSO4) 12 mg/L Iron(II) sulfate (FeSO4) 684 μg/L Monopotassium phosphate (KH2PO4) 11.6 g/L Potassium hydroxide (KOH) 3.6 g/L Magnesium sulfate (MgSO4) 171 μg/L D-(+)-glucose 20 g/L Biotin 20 μg/L Pantothenic acid 400 μg/L Inositol 1 mg/L Pyridoxine 400 μg/L Thiamine 400 μg/L Adenine 42.7 mg/L Arginine–HCl 17.1 mg/L Aspartic acid 100 mg/L Glutamic acid 85.5 mg/L Histidine 42.73 mg/L Isoleucine 25.64 mg/L Lysine–HCl 25.64 mg/L Methionine 17.1 mg/L Phenylalanine 21.4 mg/L Serine 320.4 mg/L Threonine 192 mg/L Tyrosine 25.7 mg/L See Subheading 2.4 for preparation steps 32 Tingting Xu et al.
- 46. 2. Prepare 10 g/L L-tyrosine, 4 g/L L-aspartic acid, and 24 g/L L-threonine solutions with HPLC water. Filter-sterilize each into separate baked glass bottles and store at room temperature. 3. To make the amino acid mixture, mix 1.3 g L-histidine, 1.3 g adenine, 0.52 g L-arginine–HCl, 0.52 g L-methionine, 0.78 g L-isoleucine, 0.78 g L-lysine–HCl, 0.65 g L-phenylalanine, 2.6 g L-glutamic acid, 3.9 g L-valine, and 9.75 g L-serine with 1 L HPLC water. Sterilize by filtering through a 0.2 μm filter into a baked glass bottle and store at room temperature. 4. In a baked glass bottle, mix 13.61 g KH2PO4, 1.98 g (NH4)2SO4, 4.2 g KOH pellets, and 0.2 g MgSO4 with 953.5 mL HPLC water. Sterilize by autoclaving at 121 C for 20 min. Add 1 mL FeSO4 solution (0.8 g/L) (Subheading 2.4, step 1), 3 mL L-tyrosine solution (10 g/L) (Subheading 2.4, step 2), and 38.5 mL amino acid mixture (Subheading 2.4, step 3) to bring the total volume to 1 L. This completes the preparation of yeast base medium. 5. Prepare a 200 g/L D-(+)-glucose solution in HPLC water in a baked glass bottle. Sterilize by autoclave at 121 C for 10 min. Store at room temperature. 6. The vitamin solution contains 40 mg/L thiamine, 40 mg/L pyridoxine, 40 mg/L pantothenic acid, 200 mg/L inositol, and 2 mg/L biotin in HPLC water. Sterilize by filtering through a 0.2 μm filter into a baked glass bottle and store at 4 C. 7. To make the final YMM yeast assay medium, combine 2.5 mL CuSO4 solution (prepared in Subheading 2.4, step 1), 25 mL L-aspartic acid and 8 mL L-threonine (Subheading 2.4, step 2), 854.5 mL yeast base medium (Subheading 2.4, step 4), 100 mL glucose solution (Subheading 2.4, step 5), and 10 mL vitamin solution (Subheading 2.4, step 6) to a final volume of 1 L and filter through a 0.2 μm filter membrane into a baked glass bottle. Store at room temperature for routine use or at 4 C for long term (1 month). 3 Methods 3.1 Preparation of Environmental Samples 1. Extract 1 L aqueous sample into 1 mL HPLC-grade methanol using solid phase extraction U.S. EPA method 1694 to gener- ate a 1000 concentrate. 2. From the 1000 concentrate, follow similar dilution patterns in Subheading 2.3 (steps 2–6) to generate a full scale of sample Monitoring Emerging Contaminants Through Microbial Bioreporters 33
- 47. extract dilutions consisting of 12 concentrations ranging from 1000 to 0.25. 3. Store sample methanol dilutions at 4 C. 3.2 Yeast Bioassay 1. Thaw a 1 mL-vial frozen stock (80 C) of strain BLYES, BLYAS, and BLYR at room temperature (see Note 3). 2. Immediately following thawing, inoculate the cells into 30 mL YMM without leucine, uracil, or tryptophan in a baked 250 mL glass flask. 3. Incubate at 28 C and 200 rpm shaking to an approximate optical density at 600 nm (OD600) of 0.5–0.8 (see Note 4). 4. Pipet 20 μL of each of the 18 E2 or DHT standards into individual wells of an opaque 96-well microtiter plate for BLYES estrogenicity or BLYAS androgenicity detection, respectively (Fig. 1). 5. Add 20 μL methanol into three separate wells as negative controls for each assay (see Note 5) (Fig. 1). Fig. 1 An example 96-well plate layout for BLYES and BLYAS assays. Preload 20 μL of each standard dilution, 20 μL methanol, and 100 μL of each sample dilution to corresponding wells shown on the plate. Include three empty wells as blanks. Allow methanol to fully evaporate prior to adding 200 μL of yeast culture to each well of the plate (including the empty wells). BLYR assay is similarly plated, except that no chemical standards are needed 34 Tingting Xu et al.
- 48. 6. Add 100 μL of each of the 12 sample methanol extract dilu- tions into individual wells of an opaque 96-well microtiter plate (Fig. 1). 7. Allow the methanol to evaporate until no remaining visible wet spot. This usually takes 30 min to 1 h. 8. Add 200 μL BLYES culture into each of the wells preloaded with E2 standards and samples of interest for estrogenicity assay. Avoid bubbles. 9. Pipet 200 μL BLYAS culture into each of the wells preloaded with DHT standards and samples of interest for androgenicity assay. 10. Add 200 μL BLYR culture into each of the sample wells for toxicity monitoring. 11. Add 200 μL BLYES, BLYAS, or BLYR culture to triplicate wells preloaded with only methanol as negative controls for each assay. 12. Final assay concentrations of E2, DHT, and sample extracts after mixing with yeast culture range from 1 107 M to 2.5 1013 M, 1 106 M to 2.5 1012 M, and 500 to 0.125, respectively. 13. Add 200 μL BLYES or BLYAS cells to three additional empty wells as blanks to monitor potential contamination of the methanol solvent (see Note 6). 14. Seal the plate with a breathable-sealing membrane. 15. Transfer the plate into a microplate reader and measure biolu- minescence using a 1 s/well integration time every hour for 8 h at 28 C (see Note 7). 16. Use bioluminescence readings collected 4–8 h post treatment for analysis (see Note 8). 3.3 Calculation of IC20 for Toxicity Evaluation 1. Inhibitory concentration (IC20) is used to evaluate sample toxicity and is defined as the sample concentration capable of reducing the bioluminescence by 20% compared to methanol only controls in BLYR. Using an example dataset (Table 2) for demonstration, this section provides a step-by-step tutorial of toxicity calculation. 2. Calculate the average bioluminescence from BLYR controls exposed to only methanol. 3. Calculate the relative bioluminescence from sample-treated BLYR cells by dividing the measured bioluminescence by the calculated average methanol control readings. 4. Plot the relative bioluminescence against logarithmically trans- formed sample concentration factors (log(CF)) (Fig. 2). Monitoring Emerging Contaminants Through Microbial Bioreporters 35
- 49. Table 2 An example data set of BLYES estrogenicity, BLYAS androgenicity, and BLYR toxicity test of environmental samples Sample dilutions Concentration factor () BLYR (RLU) BLYES (RLU) BLYAS (RLU) 500 216 342 4133 250 249 209 2639 125 255 191 1514 50 287 170 707 25 295 283 857 12.5 286 265 539 5 328 222 522 2.5 352 198 549 1.25 348 187 559 0.5 337 166 521 0.25 341 168 523 0.125 331 163 501 Methanol control BLYR (RLU) BLYES (RLU) BLYAS (RLU) 347 175 526 340 190 498 341 183 496 Chemical standards [E2] (M) BLYES (RLU) [DHT] (M) BLYAS (RLU) 1 107 3934 1 106 8263 5 108 4211 5 107 8314 2.5 108 3947 2.5 107 10,713 1 108 4779 1 107 9799 5 109 4016 5 108 8248 2.5 109 3174 2.5 108 8104 1 109 2979 1 108 7921 5 1010 1608 5 109 5172 2.5 1010 842 2.5 109 3090 1 1010 414 1 109 922 5 1011 220 5 1010 516 2.5 1011 212 2.5 1010 515 1 1011 183 1 1010 565 5 1012 188 5 1011 478 2.5 1012 179 2.5 1011 477 1 1012 192 1 1011 494 5 1013 176 5 1012 482 2.5 1013 190 2.5 1012 481 RLU, relative light unit 36 Tingting Xu et al.
- 50. 5. Choose data points from the linear portion of the curve and fit the data with a linear regression model: Relative bioluminescence ¼ intercept þ slope log CF ð Þ In this example, data points for the four highest concentrations are used for analysis. A linear relationship between relative biolumines- cence and log(CF) is predicted as: Relative bioluminescence ¼ 1:1641 0:1932 log CF ð Þ Calculate IC20 with a relative bioluminescence value of 0.8 and other parameters in the predicted linear model. The IC20 of the sample in this example is calculated to be 76.7, meaning that the sample needs to be concentrated 76.7 times to cause a 20% reduc- tion of bioluminescence in BLYR. 3.4 Calculation of Equivalent Estrogenicity and Androgenicity Sample estrogenicity and androgenicity is expressed as equivalent E2 and DHT concentration, respectively, which are calculated based on the bioreporter response to chemical standards. Using an example dataset (Table 2) for demonstration, this section pro- vides a step-by-step tutorial of equivalent estrogenicity and andro- genicity calculation. 1. Plot the bioluminescent readings against the E2 and DHT standards assay concentrations (Fig. 3). Fig. 2 BLYR toxicity assay data analysis. Relative bioluminescence is plotted against logarithmically trans- formed sample concentration factors (log(CF)) for the full scale of sample dilutions (open circle). Data points from the linear portion of the curve (closed circle) are used for linear regression prediction Monitoring Emerging Contaminants Through Microbial Bioreporters 37
- 51. 2. Fit the data points using a four-parameter logistic regression model to generate a chemical standard curve: Bioluminescence ¼ min þ max min ð Þ= 1 þ concentration=EC50 ð Þ hillslope ð Þ h i Fig. 3 BLYES and BLYAS chemical standard curve. Sigmoidal dose–response curves are predicted using a four-parameter logistic regression model for E2 (a) and DHT (b) in BLYES and BLYAS assay, respectively. RLU, relative light unit 38 Tingting Xu et al.
- 52. In this model, bioluminescence and concentration are vari- ables, whereas “min,” “max,” “EC50,” and “hillslope” are parameters that can be solved. In this example, the EC50 value of E2 in the BLYES assay and DHT in the BLYAS assay is determined to be 7.0 1010 M and 4.2 109 M, respec- tively, and the fitted standard curve can be expressed as: BLYES bioluminescence ¼ 176:5þ 4182:7176:5 ð Þ= 1þ E2 ½ = 7:01010 1:48 ð Þ h i BLYAS bioluminescence ¼ 456:1þ 9037:8456:1 ð Þ= 1þ DHT ½ = 4:2109 1:76 ð Þ h i 3. To calculate the equivalent E2 or DHT concentration in the sample, use only data points from sample concentrations that are not toxic (i.e., relative bioluminescence 0.8 as determined in the BLYR assay) and result in a bioluminescent response greater than mean + 3 standard deviation of the methanol controls in the BLYES and BLYAS assays. In this example, samples at 25, 12.5, and 5 exposure concentration meets this criteria in the BLYES assay and therefore are used to calculate the equivalent E2 concentration of the preconcen- trated (1) sample, resulting in 2.5 1012 M, 4.3 1012 M, and 6.8 1012 M, respectively. The equiva- lent E2 concentration of the 1 sample is then reported as the average of the three calculated values (3.4 1012 M). Simi- larly, the equivalent DHT concentrations of the 1 sample is calculated to be 2.1 1011 M using bioluminescence data from BLYAS cells exposed to 50 and 25 sample concentrations. 4 Notes 1. Plasticware should not be used for medium storage and yeast growth as they can leach estrogenic and/or androgenic chemi- cals during use, leading to false positive results. Baking in a muffle furnace is highly recommended for glassware steriliza- tion to remove any organic residue that might interfere with the assay. 2. The DHT standards are prepared at a different concentration range from the E2 standards due to different sensitivities between the BLYAS and BLYES strains. 3. Repeated liquid subculture is not recommended and can lead to decreased sensitivity. To make frozen stocks, inoculate an isolate grown on YMM agar without leucine, uracil, or trypto- phan into 30 mL fresh liquid medium in a baked sterile glass flask and incubate at 28 C with 200 rpm shaking to an OD600 of 1.0. Mix the yeast culture with an equal volume of 50% Monitoring Emerging Contaminants Through Microbial Bioreporters 39
- 53. sterile glycerol (prepared with HPLC water and stored in a baked sterile glass bottle). Aliquot the yeast–glycerol mixture into individual cryogenic tubes. Store at 80 C. 4. The BLYES and BLYAS strains produce optimal sensitivity to target chemical at this range of cell density and growth phase. Assays with a lower cell density may result in a delay in response time, whereas reduced sensitivity may be observed when using a stationary phase culture. 5. It is necessary to use the same aliquot of methanol that is used for standard and sample preparation as negative controls. 6. The methanol negative controls and yeast blank controls should produce similar bioluminescence. An increase in biolu- minescence in the methanol controls indicates contamination in the solvent. 7. The integration time can be adjusted from 0.1 to 1 s/well depending on instrument sensitivity. The reading intervals and total assay time can also be adjusted based on specific applications. Assays longer than 8 h are not recommended due to decreased yeast activity. 8. Induction of bioluminescence in response to E2 or DHT expo- sure can be detected in as early as 3 h. However, 4–8 h exposure is recommended to achieve optimal sensitivity. Acknowledgments Research funding was provided by the National Institute of Food and Agriculture, US Department of Agriculture, under award num- ber 2015-33610-23598 and the US National Science Foundation, Chemical, Bioengineering, Environmental and Transport Systems (CBET) program, under award number 1530953. References 1. Vogel J (2004) Tunnel vision: the regulation of endocrine disruptors. Pol Sci 37(3):277–303 2. Kabir ER, Rahman MS, Rahman I (2015) A review on endocrine disruptors and their possi- ble impacts on human health. Environ Toxicol Pharmacol 40(1):241–258. https:/ /doi.org/ 10.1016/j.etap.2015.06.009 3. Sanseverino J, Gupta RK, Layton AC, Patter- son SS, Ripp SA, Saidak L, Simpson ML, Schultz TW, Sayler GS (2005) Use of Saccha- romyces cerevisiae BLYES expressing bacterial bioluminescence for rapid, sensitive detection of estrogenic compounds. Appl Environ Microbiol 71(8):4455–4460. https:/ /doi. org/10.1128/AEM.71.8.4455-4460.2005 4. Eldridge M, Sanseverino J, Layton A, Easter J, Schultz T, Sayler G (2007) Saccharomyces cer- evisiae BLYAS, a new bioluminescent biore- porter for detection of androgenic compounds. Appl Environ Microbiol 73 (19):6012–6018 5. Gupta RK, Patterson SS, Ripp SA, Sayler GS (2003) Expression of the Photorhabdus lumi- nescens lux genes (luxA, B, C, D, and E) in Saccharomyces cerevisiae. FEMS Yeast Res 4 (3):305–313 6. Sanseverino J, Eldridge ML, Layton AC, Easter JP, Yarbrough J, Schultz TW, Sayler GS (2009) Screening of potentially hormonally active che- micals using bioluminescent yeast bioreporters. 40 Tingting Xu et al.
- 54. Toxicol Sci 107(1):122–134. https:/ /doi.org/ 10.1093/toxsci/kfn229 7. Krifaton C, Kriszt B, Risa A, Szoboszlay S, Cserháti M, Harkai P, Eldridge M, Wang J, Kukolya J (2013) Application of a yeast estro- gen reporter system for screening zearalenone degrading microbes. J Hazard Mater 244:429–435. https:/ /doi.org/10.1016/j. jhazmat.2012.11.063 8. Osimitz T, Eldridge M, Sloter E, Welsh W, Ai N, Sayler G, Menn F, Toole C (2012) Lack of androgenicity and estrogenicity of the three monomers used in Eastman’s Tritan copolye- sters. Food Chem Toxicol 50(6):2196–2205 9. Balazs A, Krifaton C, Risa A, Cserhati M, Kukolya J, Toth A, Hahn J, Szoboszlay S, Eldridge M, Wang J, Kriszt B (2014) Biodeg- radation of 5 alpha-dihydrotestosterone to non-androgenic products. Int Biodeterior Bio- degradation 93:162–167. https:/ /doi.org/10. 1016/j.ibiod.2014.05.008 10. Bergamasco AMD, Eldridge M, Sanseverino J, Sodre FF, Montagner CC, Pescara IC, Jardim WF, Umbuzeiro GD (2011) Bioluminescent yeast estrogen assay (BLYES) as a sensitive tool to monitor surface and drinking water for estrogenicity. J Environ Monit 13 (11):3288–3293. https:/ /doi.org/10.1039/ c1em10464k 11. Jardim W, Montagner C, Pescara I, Umbuzeiro G, Bergamasco A, Eldridge M, Sodre F (2012) An integrated approach to eval- uate emerging contaminants in drinking water. Sep Purif Technol 84:3–8. https:/ /doi.org/ 10.1016/j.seppur.2011.06.020 12. Ruan T, Liang D, Song SJ, Song MY, Wang HL, Jiang GB (2015) Evaluation of the in vitro estrogenicity of emerging bisphenol analogs and their respective estrogenic contributions in municipal sewage sludge in China. Chemo- sphere 124:150–155. https:/ /doi.org/10. 1016/j.chemosphere.2014.12.017 13. Wang J, Eldridge M, Menn FM, Dykes T, Say- ler G (2015) Standardized application of yeast bioluminescent reporters as endocrine disrup- tor screen for comparative analysis of wastewa- ter effluents from membrane bioreactor and traditional activated sludge. Ecotoxicology 24 (10):2088–2099. https:/ /doi.org/10.1007/ s10646-015-1556-z Monitoring Emerging Contaminants Through Microbial Bioreporters 41
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- 56. examined the diamond; it was of the finest water. “How much do you ask for it?” said he. “Two hundred crowns,” said Massaccio, thinking his demand to be great; it was hardly the tenth part of the value of the stone. The jeweller looked at Massaccio, and said: “To sell it at that price you must be a robber, and I arrest you!” “If it is not worth so much, give me less,” said Massaccio; “I am not a robber, I am an honest man; it was the serpent who gave me the diamond.” But the police now arrived and conducted him before the magistrate. There he recounted his adventure, which appeared to be a mere fairy vision. Yet as the Signor Vitalis was implicated in the story, the magistrate referred the affair to the state inquisition, and Massaccio appeared before it. “Relate to us your history,” said one of the inquisitors, “and lie not, or we will have you thrown into the canal.” Massaccio related his adventure. “So,” said the inquisitor, “you saved the Signor Vitalis?” “Yes, noble signors.” “And he promised you a marriage portion for your bride, and his palace at Venice for yourself?” “Yes, noble signors.” “And he drove you like a beggar from his door?” “Yes, noble signors.” “Let the Signor Vitalis appear,” said the same inquisitor. Vitalis appeared. “Do you know this man, Signor Vitalis?” said the inquisitor. “No, I know him not,” replied Vitalis. The inquisitors consulted together. “This man,” said they, speaking of Massaccio, “is evidently a knave and a cheat; he must be thrown into prison. Signor Vitalis, you are acquitted.” Then, making a sign to an officer of police, “Take that man,” said he, “to prison.” Massaccio fell on his knees in the middle of the hall. “Noble signors, noble signors,” said he, “it is possible that the diamond may have been stolen; the serpent who gave it me may have wished to
- 57. deceive me. It is possible that the ape, the lion, and the serpent may all be an illusion of the demon, but it is true that I saved the Signor Vitalis. Signor Vitalis” (turning to him), “I ask you not for the marriage portion for my bride, nor for your palace of marble, but say a word for me; suffer me not to be thrown into prison; do not abandon me; I did not abandon you when you were in the pit.” “Noble signors,” said Vitalis, bowing to the tribunal, “I can only repeat what I have already said: I know not this man. Has he a single witness to produce?” At this moment the whole court was thrown into fear and astonishment, for the lion, the monkey, and the serpent, entered the hall together. The monkey was mounted on the back of the lion, and the serpent was twined round the arm of the monkey. On entering, the lion roared, the monkey spluttered, and the serpent hissed. “Ah! these are the animals of the pit,” cried Vitalis, in alarm. “Signor Vitalis,” resumed the chief of the inquisitors, when the dismay which this apparition had caused had somewhat diminished, “you have asked where were the witnesses of Massaccio. You see that God has sent them at the right time before the bar of our tribunal. Since, then, God has testified against you, we should be culpable before Him if we did not punish your ingratitude. Your palace and your possessions are confiscated, and you shall pass the rest of your life in a narrow prison. And you,” continued he, addressing himself to Massaccio, who was all this time caressing the lion, the monkey, and the serpent, “since a Venetian has promised you a palace of marble, and a portion for your bride, the republic of Venice will accomplish the promise; the palace and possessions of Vitalis are thine. You,” said he to the secretary of the tribunal, “draw up an account of all this history, that the people of Venice may know, through all generations, that the justice of the tribunal of the state inquisition is not less equitable than it is rigorous.” Massaccio and his wife lived happily for many years afterwards in the palace of Vitalis with the monkey, the lion, and the serpent; and Massaccio had them represented in a picture, on the wall of his palace, as they entered the hall of the tribunal, the lion carrying the monkey, and the monkey carrying the serpent. “To what source can this tale be traced?”
- 58. “To the Arabian fable book called Callah-u-Dumnah,” replied Lathom. “Mathew Paris recites it as a fable commonly used by our crusading Richard to reprove his ungodly nobles, and old Gower has versified it in his Confessio Amantis. The translator in Blackwood seems not to have been aware of its existence in the Gesta Romanorum, content to translate it from the later version of Massenius, a German Jesuit, who lived at Cologne in 1657.” “Few subjects,” said Herbert, “seem more involved than the history of didactic fiction. The more mysterious an investigation bids fair to be, the less we have to depend on fact, and the more we are at the mercy of conjecture, so much the more does the mind love to grasp at the mystery, and delight in the dim perspective and intricacies of the way. Each successive adventurer finds it more easy to pull down the various bridges, and break in the various cuttings by which his predecessor has endeavored to make the way straight, than to throw his own bridge over the river or the morass of time that intervenes between the traveller and the goal.” “Four distinct sources,” said Lathom, “have been contended for: the Scandinavian bards, the Arabians of the Spanish peninsula, the Armoricans or Bretons, and the classical authors of Greece and Rome. Mallet and Bishop Percy came forward as the advocates of Scandinavia; Dr. Wharton writes himself the champion of the Spanish Arabians; Wilson is rather inclined to the Breton theory; and Dr. Southey and Mr. Dunlop come forward as the advocates of the classical and mythological authors; whilst Sir Henry Ellis would reconcile all differences by a quiet jumble of Breton scenes colored by Scandinavia and worked by Arabian machinery. Let us, however, adjourn this subject until to-morrow, as I wish to read you another of these tales, in order to give you some idea of the moral applications and explanations appended to them by the monkish writers. We will take Jovinian the Proud Emperor, and in this case you must be content with my own translation.”
- 59. JOVINIAN THE PROUD EMPEROR. In the days of old, when the empire of the world was in the hands of the lord of Rome, Jovinian was emperor. Oft as he lay on his couch, and mused upon his power and his wealth, his heart was elated beyond measure, and he said within himself: “Verily, there is no other god than me.” It happened one morning after he had thus said unto himself, that the emperor arose, and summoning his huntsmen and his friends, hastened to chase the wild deer of the forest. The chase was long and swift, and the sun was high in the heavens, when Jovinian reined up his horse on the bank of a clear bright stream that ran through the fertile country on which his palace stood. Allured by the refreshing appearance of the stream, he bade his attendants abide still, whilst he sought a secluded pool beneath some willows, where he might bathe unseen. The emperor hastened to the pool, cast off his garments, and revelled in the refreshing coolness of the waters. But whilst he thus bathed, a person like to him in form, in feature, and in voice, approached the river’s bank, arrayed himself unperceived in the imperial garments, and then sprang on Jovinian’s horse, and rode to meet the huntsmen, who, deceived by the likeness and the dress, obeyed his commands, and followed their new emperor to the palace gates. Jovinian at length quitted the water, and sought in every direction for his apparel and his horse, but could not find them. He called aloud upon his attendants, but they heard him not, being already in attendance on the false emperor. And Jovinian regarded his nakedness and said: “Miserable man that I am! to what a state am I reduced! Whither shall I go? Who will receive me in this plight? I bethink me there is a knight hereabout whom I have advanced to great honor; I will seek him, and with his assistance regain my palace, and punish the person who has done me this wrong.” Naked and ashamed, Jovinian sought the gate of the knight’s castle, and knocked loudly at the wicket.
- 60. “Who art thou, and what dost thou seek?” asked the porter, without unclosing the gate. “Open, open, sirrah!” replied the emperor, with redoubled knocks on the wicket. “In the name of wonder, friend, who art thou?” said the old porter as he opened the gate, and saw the strange figure of the emperor before the threshold. “Who am I, askest thou, sirrah? I am thy emperor. Go, tell thy master, Jovinian is at his gate, and bid him bring forth a horse and some garments, to supply those that I have been deprived of.” “Rascal,” rejoined the porter—“thou the emperor! Why, the emperor but just now rode up to the castle, with all his attendants, and honored my master by sitting with him at meat in the great hall. Thou the emperor! a very pretty emperor indeed; faugh, I’ll tell my master what you say, and he will soon find out whether you are mad, drunk, or a thief.” The porter, greatly enraged, went and told his lord how that a naked fellow stood at the gate, calling himself the emperor, and demanding clothes and a good steed. “Bring the fellow in,” said the knight. So they brought in Jovinian, and he stood before the lord of the castle, and again declared himself to be the emperor Jovinian. Loud laughed the knight to the emperor. “What, thou my lord the emperor! art mad, good fellow? Come, give him my old cloak; it will keep him from the flies.” “Yes, sir knight,” replied Jovinian, “I am thy emperor, who advanced thee to great honor and wealth, and will shortly punish thee for thy present conduct.” “Scoundrel!” said the knight, now enraged beyond all bounds, “traitor! thou the emperor! ay, of beggars and fools. Why, did not my lord but lately sit with me in my hall, and taste of my poor cheer? and did not he bid me ride with him to his palace gate, whence I am but now returned? Fool, I pitied thee before; now I see thy villany. Go, turn the fellow out, and flog him from the castle-ditch to the river- side.”
- 61. And the people did as the knight commanded them. So when they ceased from flogging the emperor, he sat him down on the grass, and covered him with the tattered robe, and communed on his own wretchedness. “Oh, my God!” said Jovinian,—for he now thought of other gods but himself,—“is it possible that I have come to such a state of misery, and that, through the ingratitude of one whom I have raised so high!” And as he thus spake, he thought not of his own ingratitude to his God, through whom alone all princes reign and live. And now he brooded over vengeance—“Ay,” said he, as he felt the sore weals on his back from the scourging; “ay, I will be avenged. When he next sees me, he shall know that he who gives can also take away. Come, I will seek the good duke, my ablest counsellor; he will know his sovereign, and gladly aid him in his calamity.” And with these thoughts he wrapped his cloak round him, and sought the house of the good duke. Jovinian knocked at the gate of the duke’s palace, and the porter opened the wicket, and seeing a half-naked man, asked him why he knocked, and who he was. “Friend,” replied the emperor, “I am Jovinian. I have been robbed of my clothes whilst bathing, and am now with no apparel, save this ragged cloak, and no horse; so tell the duke the emperor is here.” The porter, more and more astonished at the emperor’s words, sought his master, and delivered Jovinian’s message to him. “Bring in the poor man,” said the duke; “peradventure he is mad.” So they brought Jovinian unto the duke’s great hall, and the duke looked on him, but knew him not. And when Jovinian reiterated his story, and spoke angrily unto the duke, he pitied him. “Poor mad fellow,” said the good duke, “I have but just now returned from the palace, where I left the very emperor thou assumest to be. Take him to the guard-house. Perhaps a few days’ close confinement on bread and water may cool his heated brain. Go, poor fellow; I pity thee!” So the servants did as their lord commanded, and they fed Jovinian on bread and water, and after a time turned him out of the castle; for he still said he was the emperor. Sorely and bitterly did the emperor weep and bewail his miserable fate when the servants drove him from the castle gate. “Alas, alas!”
- 62. he exclaimed in his misery, “what shall I do, and whither shall I resort? Even the good duke knew me not, but regarded me as a poor madman. Come, I will seek my own palace, and discover myself to my wife. Surely she will know me at least.” “Who art thou, poor man?” asked the king’s porter of him when he stood before the palace gate and would have entered in. “Thou oughtest to know me,” replied Jovinian, “seeing thou hast served me these fifteen years.” “Served you, you dirty fellow,” rejoined the porter. “I serve the emperor. Serve you, indeed!” “I am the emperor. Dost thou not know me? Come, my good fellow, seek the empress, and bid her, by the sign of the three moles on the emperor’s breast, send me hither the imperial robes, which some fellow stole whilst I was bathing.” “Ha! ha! fellow; well, you are royally mad. Why, the emperor is at dinner with his wife. Well, well, I’ll do thy bidding, if it be but to have the whipping of thee afterwards for an impudent madman. Three moles on the emperor’s breast! how royally thou shalt be beaten, my friend.” When the porter told the empress what the poor madman at the gate had said, she held down her head, and said, with a sorrowful voice, unto her lord: “My good lord and king, here is a fellow at the palace gate that hath sent unto me, and bids me, by those secret signs known only to thee and me, to send him the imperial robes, and welcome him as my husband and my sovereign.” When the fictitious emperor heard this, he bade the attendants bring in Jovinian. And lo, as he entered the hall, the great wolf- hound, that had slept at his feet for years, sprang from his lair, and would have pulled him down, had not the attendants prevented him; whilst the falcon, that had sat on his wrist in many a fair day’s hawking, broke her jesses, and flew out of the hall: so changed was Jovinian the emperor. “Nobles and friends,” said the new emperor, “hear ye what I will ask of this man.” And the nobles bowed assent, whilst the emperor asked Jovinian his name, and his business with the empress.
- 63. “Askest thou me who I am, and wherefore I am come?” rejoined Jovinian. “Am not I thy emperor, and the lord of this house and this realm?” “These our nobles shall decide,” replied the new king. “Tell me now, which of us twain is your emperor?” And the nobles answered with one accord: “Thou dost trifle with us, sire. Can we doubt that thou art our emperor, whom we have known from his childhood? As for this base fellow, we know not who he is.” And with one accord the people cried out against Jovinian that he should be punished. On this the usurper turned to the empress of Jovinian—“Tell me,” said he, “on thy true faith, knowest thou this man who calls himself emperor of this realm?” And the empress answered: “Good my lord, have not thirty years passed since I first knew thee, and became the mother of our children? Why askest thou me of this fellow? and yet it doth surprise me how he should know what none save you and I can know?” Then the usurper turned to Jovinian, and with a harsh countenance rebuked his presumption, and ordered the executioners to drag him by the feet by horses until he died. This said he before all his court; but he sent his servant to the tailor, and commanded him to scourge Jovinian; and for this once to set him free. The deposed emperor desired death. “Why,” said he to himself, “should I now live? my friends, my dependents, yea, even the partner of my bed shuns me, and I am desolate among those whom my bounties have raised. Come, I will seek the good priest, to whom I so often have laid open my most secret faults: of a surety, he will remember me.” Now the good priest lived in a small cell, nigh to a chapel about a stone’s-cast from the palace gate; and when Jovinian knocked, the priest, being engaged in reading, answered from within: “Who is there? why troublest thou me?” “I am the emperor Jovinian; open the window, I would speak to thee,” replied the fugitive. Immediately the narrow window of the cell was opened, and the priest, looking out, saw no one save the poor half-clothed Jovinian.
- 64. “Depart from me, thou accursed thing!” cried the priest; “thou art not our good lord the emperor, but the foul fiend himself, the great tempter.” “Alas, alas!” cried Jovinian, “to what fate am I reserved, that even my own good priest despises me! Ah me, I bethink me—in the arrogance of my heart, I called myself a god: the weight of my sin is grievous unto me. Father, good father, hear the sins of a miserable penitent.” Gladly did the priest listen to Jovinian; and when he had told him all his sins, the good priest comforted the penitent, and assured him of God’s mercy, if his repentance was sincere. And so it happened that on this a cloud seemed to fall from before the eyes of the priest; and when he again looked on Jovinian he knew him to be the emperor, and he pitied him, clothing him with such poor garments as he had, and went with him to the palace gate. The porter stood in the gateway, and as Jovinian and the priest drew near he made a lowly obeisance, and opened the gate for the emperor. “Dost thou know me?” asked the emperor. “Very well, my lord,” replied the servant; “but I wish that you had not left the palace.” So Jovinian passed on to the hall of his palace; and as he went, all the nobles rose and bowed to the emperor; for the usurper was in another apartment, and the nobles knew again the face of Jovinian. But a certain knight passed into the presence of the false emperor. “My lord,” said he, “there is one in the great hall to whom all men bow, for he so much resembleth you that we know not which is the emperor.” Then said the usurper to the empress: “Go and see if you know this man.” “Oh, my good lord,” said the empress, when she returned from the hall, “whom can I believe? are there, then, two Jovinians?” “I will myself go and determine,” rejoined the usurper, as he took the empress by her hand, and, leading her into the great hall, placed her on the throne beside himself. “Kinsfolk and nobles,” said the usurper, “by the oaths ye have sworn, determine between me and this man.”
- 65. And the empress answered: “Let me, as in duty bound, speak first. Heaven be my witness, I know not which is my lord and husband.” And all the nobles said the same. Thereupon the feigned Jovinian rose and spake: “Nobles and friends, hearken! that man is your emperor and your master; hear ye him; know that he did exalt himself above that which was right, and make himself equal unto God. Verily he hath been rewarded; he hath suffered much indignity and wrong, and, of God’s will, ye knew him not; he hath repented him of his grievous sin, and the scourge is now removed; he has made such satisfaction as man can make. Hear ye him, know him, obey him.” As the feigned emperor thus addressed the astonished nobles, his features seemed illumined with a fair and spiritual light, his imperial robes fell from off him, and he stood confessed before the assembly an angel of God, clothed in white raiment. And, as he ended his speech, he bowed his head, and vanished from their sight. Jovinian returned to his throne, and for three years reigned with so much mercy and justice, that his subjects had no cause to regret the change of their emperor. And it came to pass, after the space of three years, the same angel appeared to him in a dream, and warned him of his death. So Jovinian dictated his troublous life to his secretaries, that it might remain as a warning unto all men against worldly pride, and an incitement to the performance of our religious duties. And when he had so done, he meekly resigned himself, and fell asleep in death. “So much for the story, as a story; now for the moral, with all that eccentric spirit of refinement and abstraction with which the age was characterized,” said Herbert. “The moral in this case is less eccentric than in many to which I hope we shall come before Christmas is over.” “Jovinian was but the picture of the proud, worldly-minded man, entirely given up to vanity and folly. The first knight whose castle he visited was True Wisdom, ever disdainful of the pomps and vanities of the world. The next knight was Conscience. The dog that turned against his old master, was the lusts of the flesh, our own evil desires, which will ever in the end turn against those who have pampered them. The falcon is God’s grace; the empress, man’s soul; and the clothes in which the good priest clothed the half-frozen emperor, are those kingly virtues which he had thrown off, when he gave loose rein to the vanities of the world.”
- 66. “It must be admitted,” remarked Herbert, “that from very early times a secondary meaning was commonly attached to every important work; it progressed from the sacred writings through the poetic fictions of the classics, to compositions professedly allegorical. The want of discrimination, which in our eyes assumes much of the appearance of profane levity, with which the fictions of the classics were interpreted to signify the great truths and mysteries of religion, was, perhaps, hardly reprehensible in the simple state of knowledge which prevailed at the time when these attempts at secondary interpretation were made.” “And hence it was,” said Lathom, “that in the early ages it might seem to partake of little levity to prefigure our Saviour’s birth in that of Bacchus; his sufferings and death in that of Actæon, or his resurrection in the legend of Hercules, as related by Lycophron; as late as the thirteenth century the Franciscan Walleys wrote a moral and theological exposition of the Metamorphoses of Ovid.” “But surely the writers of that age did not stop there,” said Thompson; “was it not the case, that to these expositions succeeded compositions professedly allegorical, and which the spirit of refinement of that age resolved into further allegories, for which they were never intended?” “Undoubtedly so!” replied Lathom; “it was not enough that the writer of the ‘Romaunt of the Rose’ had allegorized the difficulties of an ardent lover in the accomplishment of his object, under the mystery of the rose which was to be gathered in a fair but almost inaccessible garden. Every profession saw in this allegory the great mystery of their craft. To the theologian it was the rose of Jericho, the New Jerusalem, the Blessed Virgin, or any other mystery to which obstinate heretics were unable to attain; to the chemist it was the philosopher’s stone; to the lawyer it was the most consummate point of equity; to the physician the infallible panacea, the water of life; and does not this spirit of allegory extend to the present day, only in a somewhat different form?” “Not unlike the present system of commentating,” remarked Henry Herbert. “As soon as a poet has attained to any great reputation, and death has sealed up his writings, then comes the host of annotators and critics, each one more intent than his predecessor to develop the mind of the writer, to discover with what hidden intentions, with what feelings, this or that passage was written, and to build on some stray expression a mighty theory, for some more clever writer to overthrow, and raise a new fabric on its ruins. And in these attempts it is not the old author whose glory is sought to be heightened, but the new man who would ascend the ladder of reputation on the labors of the ‘man of old.’” “Far different,” rejoined Lathom, “was the spirit which prompted the fashion of resolving every thing into allegories in the middle ages; nor, indeed, is it to be solely charged to an unmeaning and wanton spirit of refinement. ‘The same apology,’ says Wharton, ‘may be offered for cabalistic interpreters, both of the classics and of the old romances. The former, not willing that those books should be quite exploded which contained the ancient mythology, labored to reconcile the apparent absurdities of the pagan system with the Christian mysteries, by demonstrating a figurative resemblance. The latter, as true learning began to
- 67. dawn, with a view of supporting for a time the expiring credit of giants and magicians, were compelled to palliate those monstrous incredibilities, by a bold attempt to unravel the mystic web which had been woven by fairy hands, and by showing that truth was hid under the gorgeous veil of gothic invention.’ And now, Thompson, we must adjourn, you to your real Greeks and Romans, Herbert and I to Aristotle’s Summum Bonum.”
- 68. “S CHAPTER II. Discussion on the Source of Fiction Renewed—The King and the Glutton— Guido, the Perfect Servant—The Middle-Age Allegories—Pliny and Mandeville’s Wonders Allegorized. urely,” said Henry Herbert, when the friends were again assembled, “surely the poems of the northern Scalds, the legends of the Arabians of Spain, the songs of the Armoricans, and the classics of the ancient world, have been the sources of the most prevalent fictions.” “The sources from which the monks themselves compiled these stories, but by no means the original sources,” replied Lathom. “The immediate source must be sought in even earlier times and more eastern climes. In some instances perverted notions of Scripture characters furnished the supernatural agency of the legend; in the majority the machinery came direct from the East, already dilated and improved. In many parts of the old Scriptures we learn how familiar the nations of the East were with spells; and the elevation of Solomon Daoud to the throne of the Genii and to the lordship of the Talisman, proves the traditional intercourse between God’s own people and the nations of the far East.” “The theory is probable,” said Thompson. “We can easily conceive how the contest of David and Goliath may have formed the foundation of many a fierce encounter between knight and giant, and the feats of Samson been dilated into the miracles of the heroes of chivalry.” “There is one very pertinent instance of such a conversion in this very book. In the Book of Tobit, which is indeed referred to in the application of the tale of ‘The Emperor Vespasian and the Two Rings,’ we find an angel in the place of a saint, enchantments, antidotes, distressed damsels, demons, and nearly all the recognized machinery of fiction. The vagaries of the Talmud, clearly derived from Eastern sources, were no small treasure on which to draw for wonders and miracles. And when we find all the machinery of the East in the poems of the Scalds, we cannot but perceive how much more reasonable it is to suppose the cold conceptions of the Northern bards to have been fed from the East, than the warm imaginations of the East to have drawn their inspiration from the North.” “Very plausible, Lathom,” replied Herbert; “but still this objection must not be neglected—the ignorance and misrepresentation of the religions of the East, shown through every page of the popular legends of the chivalric age.”
- 69. “An objection of apparent weight, I will admit; and yet may it not have been the aim of the Christian writers to represent the infidels in the worst possible light, to pervert their creed, to exaggerate their vices? The charge of idolatry, and the adoration of the golden image of Mahomet, may have been mere pious frauds.” “Admitting even this apology,” rejoined Herbert, “the difference of religion in the East and North seems another objection. The Romans adopted the legends of Greece, and naturalized them. With the mythology came the religious rites appendant to it. How did it happen that the Scalds adopted the one without falling into the other error?” “Are the cases similar?” replied Lathom; “were the nations alike? Was there no difference of predisposition in the Romans and the Scalds as to the adoption of the mythologies of the East and Greece? Had not long intercourse in the one case prepared the Romans to receive? did it not agree with their preconceived notions? Such was not the case with the Northern nations. Children, and rude children of nature, they were in no way prepared for a similar effect; but, seizing on the prominent features of the legends presented to them, they engrafted them on their own wild and terrible stories, adding to the original matter in some cases, and rejecting portions of it in others.” “Well, I will not carry this discussion further,” said Herbert, “for fear of losing a story to-night; but I by no means give up my sources of didactic fictions.” “Well, then, a truce for this evening. I will read the tale of The King and the Glutton, by which the old monk wished to illustrate the moral, that men are blinded by their own avarice.”
- 71. THE KING AND THE GLUTTON. There once lived a king of Rome, who, out of charity to the blind, decreed that every subject of his that was so afflicted, should be entitled to receive a hundred shillings from the royal treasury. Now there was in Rome a club of men who lived for the world alone, and spent all they had in rioting and eating. Seven days had they continued revelling in one tavern, when the host demanded to be paid his bill. Every one searched his pockets, but still there was not enough to pay the reckoning. “There still wants one hundred shillings,” said the innkeeper; “and until that is paid, ye go not hence.” These young men knew not what to do, as they were penniless. “What shall we do?” said they one to another. “How can we pay so large a sum?” At length one bethought him of the king’s edict. “Listen,” said he, “listen to me; does not the king give one hundred shillings to every blind man that applies for it?” “Even so,” said the rest; “but what then? we are not blind.” “What then?” rejoined the young man. “Come, let us cast lots who shall be made blind, that when he is deprived of sight we may take him to the king’s palace, and obtain the hundred shillings.” So the young men cast lots, and the lot fell upon the man who had proposed this plan. And the rest took him, and putting out his eyes, led him to the king’s palace. When they knocked at the gate, the porter opened the wicket, and demanded their business. “Business,” said they; “see ye not our companion is blind? he seeks to receive the king’s benevolent gift.” “The blindness is rather sudden,” muttered the porter, who knew the young man by sight. “Well, well, I will fetch the almoner.” So the almoner, who distributed the king’s charity, came to the gate, and looking on the young man, asked him what he wanted. “A hundred shillings, which my lord the king gives to those that are blind,” replied the youth.
- 72. “Thy blindness is very sudden,” rejoined the almoner; “when did it happen, and where? for I saw thee yesterday with both eyes perfect in the tavern by the city wall.” “Last night, noble sir,” replied the blind man, “last night at that tavern I became blind.” “Go fetch the host,” said the almoner sternly, “we will look into this matter more fully.” So when the innkeeper came, he inquired of him how the matter was; and when he had heard all their deeds, he turned to the young man, and said— “Of a surety thou knowest but half the law, and dost interpret it wrong; to such as are blind by God’s act, does our gracious king give his charity; such the law protects and relieves. But thou—why art thou blind? Thinkest thou that thou dost deserve to be rewarded for voluntarily surrendering thine eyes, in order to discharge the debt thou and thy companions had contracted by gluttony and rioting? Begone, foolish man: thy avarice hath made thee blind.” So they drove away the young men from the king’s gate, lamenting their folly and wickedness. “There can be little doubt,” said Herbert, “what moral the author of this tale intended to teach. The king’s gift clearly illustrates God’s reward for forgiveness, to those that by natural infirmity and temptation fall into sin; as the withholding it from the glutton, is meant to teach us how difficult it will be to obtain the forgiveness of voluntary sin, done out of pure wickedness.” “You have found out the monk’s moral rightly in this tale, Henry; but I think you will not be so successful in that which I now propose reading to you—the story of
- 74. “GUIDO, THE PERFECT SERVANT.” There was once a great emperor of Rome named Valerius, who would that every man, according to his wishes, should serve him; so he commanded that whosoever should strike three times on the gate of his palace should be admitted to do him service. In the emperor’s kingdom was also a poor man named Guido, who, when he heard of his lord’s commands, thus spake with himself: “Now, I am a poor man, and lowly born; is it not better to live and serve than to starve and be free?” So he went to the king’s gate, and knocked three knocks; and lo, it was opened to him, according as it had been said; and he was brought before the emperor. “What seek you, friend?” asked Valerius, as Guido bowed before him. “To serve my king,” was Guido’s reply. “What service can you perform for me?” rejoined the emperor. “Six services can I perform, O king: as your body-guard, I can prepare your bed and your food, and attend your chamber. I can sleep when others watch, and watch while others sleep. As your cup- bearer, I can drink good wine, and tell whether it be so or not. I can summon the guests to my master’s banquet, to his great honor and benefit. I can kindle a fire which shall warm all that seek it, and yet not smoke. And I can show the way to the Holy Land, to the health of such as shall go thither.” “By my truth,” rejoined the emperor, “these are great things that thou dost promise. See that thou do them. Each for one year. Serve me first as my body-guard.” Guido was content to obey the emperor; and he prepared to perform his duties as his body-guard. Every night he made ready the emperor’s bed, and prepared his apparel. Every night he lay before the emperor’s chamber-door, armed at all points; whilst by his side watched a faithful dog to warn him of the approach of danger. In every thing did he minister so faithfully to his lord, that the emperor was well pleased with him, and after his first year, made him
- 75. seneschal of his castle and steward of his household. Then did Guido commence his labors in his second office. During the entire summer he gathered large stores of every thing needful into the castle, and collected much provision at little cost, by carefully watching his opportunities. Anon came on the winter, and when those who had slept during the times of plenty began to labor and lay up in their store-houses, Guido remained at ease, and completed his second year’s service with credit to himself. And now the third year of Guido’s service came on; and the emperor called for his chief butler, and said: “Mix in a cup good wine, must, and vinegar, and give it to Guido to drink; that we may know how he doth taste good drink, and what he knoweth of its qualities.” So the butler did as he was ordered, and gave the cup to Guido, who, when he had tasted of it, said: “Of a truth it was good, it is good, and it will be good.” And when the emperor asked him how these things could be, he said: “The vinegar was good, the old wine is good, and the must will be good when it is older.” So the emperor saw that he had answered rightly and discreetly of the mixture, which he knew not of before. “Go, therefore,” said Valerius, “through my country, and invite my friends to a banquet at the festival of Christmas now at hand”; and Guido bowed assent, and departed on his way. But Guido did not execute his lord’s commands—going not unto his friends, but unto his enemies. So that when the emperor descended into his banquet-hall his heart was troubled; for his enemies sat round his table, and there was not a friend among them. So he called Guido, and spake angrily to him. “How, sir! didst thou not tell me that thou knewest whom to invite to my banquet?” And Guido said: “Of a surety, my lord.” “Did not I bid thee invite my friends? and how, then, hast thou summoned all mine enemies?” And Guido said: “May thy servant speak?” So the emperor said: “Speak on.” And the servant said: “My lord, there is no season or time that thy friends may not visit thee, and be received with pleasure and honor; but it is not so with thine enemies. Then I said to myself:
- 76. ‘Conciliation and kindness would go far to convert enemies into friends.’” Now it turned out as Guido hoped; for ere the feast was ended, the king and his enemies were reconciled to each other, and became friends even unto the end of their days. So the emperor called Guido, and said: “With God’s blessing, thy design has prospered. Come, now, make for my reconciled enemies and me a fire that shall burn without smoke.” And Guido answered: “It shall be done as thou hast required, O king.” So he sent and gathered much green wood, and dried it in the sun until it was quite dry, and therewith made a fire that did cast out much heat, and yet did not smoke. So that the emperor and his friends rejoiced greatly therein. And so it was when the emperor saw how well Guido had performed his five ministries, he bade him execute his sixth service—that he might attain to great honor in his kingdom. “My lord,” said Guido, “he that would know the way to the Holy Land must follow me to the sea-shore.” So a proclamation went forth from the king to that effect; and great multitudes of men and women flocked to the sea-shore after Guido. When the people were come, Guido said: “My friends, do ye see in the ocean the things that I see?” And the people answered: “We know not.” “See ye in the midst of the waves a huge rock?” And the people answered: “It is even so. Why ask you this of us?” “Know ye all,” replied Guido, “that on that rock liveth a bird, that sitteth continually on her nest, in which are seven eggs. While she so sitteth, behold the sea is calm, and men may pass to and fro over the wide waters in safety. But when she doth quit her nest, the winds blow, and the waves rise, and many perish on the waters.” Then said the people: “How shall we know when this bird quitteth her nest?” And Guido answered: “She sitteth always, unless a sudden emergency happen; and then when she is away there cometh another bird, great and strong, that defileth her nest and breaketh her seven
- 77. eggs, which, when the first bird seeth, she flieth away, and the winds and storms arise; then must the shipman remain in port.” Then said the people: “Master, how may we prevent these things, and defend the bird and her nest from her enemy?” And Guido said: “The enemy hateth the blood of the lamb, and cannot come where that is. Sprinkle, therefore, the inside and outside of the nest with this blood; and so long as one drop remaineth the friendly bird will sit in peace, and the waves will not rage and swell, and there shall be safety on the waves of the sea.” And the people did as Guido said. They took the blood of a lamb, and sprinkled the nest and the rock therewith. Then passed the emperor and all his people to the Holy Land, and returned in peace and safety. And the emperor did as he had promised unto Guido, and rewarded the perfect servant with great riches, promoting him to high honor among the people. “I confess myself conquered,” said Henry Herbert, as soon as the story was concluded. “Some points in the allegory are clear, as the way to the Holy Land, and the sprinkling of the blood of the Lamb, but the rest are beyond my discovering.” “The explanation,” said Herbert, “is undoubtedly more recondite than any we have read as yet. The great emperor is our Father in heaven; the three blows on his gate are prayer, self-denial, charity; by these three any one may become his faithful servant. Guido is a poor Christian, by baptism made his servant. His first service is to serve his God, and to prepare the heart for virtue. His second duty is to watch; ‘for he knoweth not the day nor the hour when the Son of Man cometh.’ His third task is to taste of repentance, which was good to the saints who are departed, is good to such of us as it brings to salvation, and will be good to all in the last day. The fourth duty is to invite Christ’s enemies to be his friends, and to come to the banquet of his love for he ‘came not to call the righteous, but sinners to repentance.’ The fire that burneth without smoke, is the fire of charity, which burneth free of all ill-will and bad feeling. The way to the Holy Land is our course heavenward. We are to sail over our sea, the world; in the midst of which standeth our rock, even our heart, on which the holy bird of God’s Spirit resteth. The seven eggs are the gifts of the Spirit. When the Spirit leaves us, the Devil hasteth to defile our hearts; but the blood of the Lamb which was slain for us, even our Saviour, will ward off the attack of our enemy, so long as we are sprinkled therewith.” “The explanation is characteristic of the age,” said Herbert. “What then,” rejoined Lathom, “will you say to the moral drawn by these writers from the wonders that Pliny believed in, without seeing, and Sir John de Mandeville tried to persuade the world he believed in, from seeing?”
- 78. “What,” said Thompson, “the Anthropophagi, and men whose heads do grow beneath their shoulders?” “No creature is so monstrous, no fable so incredible, but that the monkish writers could give it a moral form, and extract from its crudities and quiddities some moral or religious lesson.” “They believed in the words of the song,” said Thompson— “‘Reason sure will always bring Something out of every thing.’” “Pliny’s dog-headed race,” said Lathom, “whom Sir John places in the island of Macumeran, and at the same time gives to them a quasi pope for a king, who says three hundred prayers per diem before he either eats or drinks, were naturally regarded by the middle-age writers as symbolical and priestly preachers of faithful hearts and frugal habits; whilst of those other islanders, who ‘have but one eye, and that in the middest of their front, and eat their flesh and fish raw,’ the monk says, ‘These be they that have the eye of prayer.’ The Astomes who have no mouths, ‘are all hairie over the whole bodie, yet clothed with soft cotton and downe, that cometh from the leaves of trees, and live only on aire, and by the smelling of sweet odors, which they draw through their nose-thrills,’ are the abstemious of this world, who die of the sin of gluttony, even as an Astome by the accidental inhalation of bad odor. Humility is signified by the absence of the head, and the placing of the face in the breast; and a tendency to sin is foreshadowed by a desire and habit of walking on all fours, or pride by short noses and goat’s feet. The Mandevillean islanders, who had flat faces without noses, and two round holes for their eyes, and thought whatsoever they saw to be good, were earth’s foolish ones; as those foul men, who have their lips so great, that when they sleep in the sun they cover all their face therewith, are the just men, the salt of the earth.” “One would as soon dream of allegorizing the Sciapodes of Aristophanes, or Homer’s Cranes and Pigmies,” said Thompson. “And so the monk has,” said Lathom. “What, the old Greek’s parasol-footed people, of whom Mandeville says with such gravity, ‘There be in Ethiope such men as have but one foot, and they go so fast that it is a great marvel; and that is a large foot, for the shadow thereof covereth the body from sun or rain, when they lie upon their back’?” “Both Aristophanes and his follower would doubtless be as surprised in learning that their sciapodes were allegorical of the charitable of this world, as Homer would in discovering in his crane-fighting pigmies those mortals who begin well but cease to do well before they attain perfection; or in their neighbors who boast of six hands, and despise clothes in favor of long hair, and live in rivers, the hardworking and laborious among men.” “The last is decidedly the most intelligible,” remarked Herbert.
- 79. “The reason of the explanation is not always clear,” replied Lathom; “it is not very easy to decide why those who have six fingers and six toes are the unpolluted, and why virtuous men are represented by a race of women with bald heads and beards flowing to their breast; nor is it very clear that virtue is well represented by a double allowance of eyes. But one curiosity remains—the beautiful men of Europe who boast a crane’s head, neck, and beak. These, says the author of the Gesta, represent judges, who should have long necks and beaks, that what the heart thinks, may be long before it reach the mouth.” “That reminds me of long Jack Bannister,” said Thompson, “who was always five minutes after every one else in laughing at a joke, as it took that extra time for it to travel from his ears to his midriff, and then back again to his mouth.” And so the evening ended with a laugh.
- 80. “A CHAPTER III. Progress of Fiction from the East to the West—The Early Christians—The Monks —The Spanish Arabians—The Crusades—The Knight and the King of Hungary—The English Gesta. dmitting the East as the immediate source of fiction,” said Henry Herbert, when they were met once more, “you must still regard the Spanish Arabians as the great disseminators of those extravagant inventions which were so peculiar to their romantic and creative genius.” “Less, perhaps, than many other sources. The absence of Moorish subjects from the earliest tales of chivalry, if it proves no more, at least shows how prevalent the tales of Charlemagne and his peers were in the eighth century, that a nation of conquerors could do little to infect them with legends of their own.” “How and when, then, Lathom, would you introduce Eastern invention?” asked Thompson. “I would refer it to much earlier ages, to the earliest of the Christian centuries, and contend that it was gradual, and therefore more natural; was the production of times and of ages, not the sudden birth and growth of one age; gradually augmenting until it attained to full and perfect stature.” “Still,” rejoined Herbert, “we want the means by which this knowledge of Eastern fable was introduced.” “Some share may be due to the return of those primitive Christians who sought refuge in the East from the persecutions of the pagan rulers of the West. Their minds were well prepared to adopt the fervent expressions of the East, and their condition prevented them from investigating the tales they heard. Hence, in the lives of these saints they were as ready to interweave the prodigies of another land, hoping, perhaps, to conciliate the minds of the Eastern Oriental to the tenets of their faith, by introducing fictitious incidents of Oriental structure, as, to conciliate the heathen, they placed their gods and goddesses in the Christian temple, dignifying them with a new name, and serving them with novel ceremonies.” “Admitting the probability, still your machinery seems deficient.” “It is but a portion of my machinery. Much more was due to the clouds of monks, who, during the third and fourth centuries, wandered over the face of the habitable world.”
- 81. “When Gibbon admits that the progress of monachism was co-extensive with that of Christianity,” suggested Frederick Thompson. “The disciples of Antony,” said Herbert, “we are assured, spread themselves beyond the tropic, over the Christian empire of Ethiopia.” “Their distribution was universal,” said Lathom; “every province, almost every city of the empire, had its ascetics; they feared no dangers, and deemed no seas, mountains, or deserts a barrier to their progress.” “The roving character of the monks, therefore,” says the last translator of the Gesta, “is another link of the chain by which I introduce Oriental fiction into the West; and it is utterly impossible (maturely weighing the habits and propensities of this class of people) that they should not have picked up and retained the floating traditions of the countries through which they passed. Some of the early romances, as well as the legends of the saints, were undoubtedly fabricated in the deep silence of the cloister. Both frequently sprung from the warmth of fancy which religious seclusion is so well tended to nourish; but the former were adorned with foreign embellishments.” “Did it ever occur to you,” said Thompson, “that the story of Ulysses and Circe bears a wondrous likeness to that of Beder the prince of Persia and Giahame princess of Samandal, and that the voyages of Sindbad afford the counterpart of the Cyclops of the Odysee?” “It would be but consistent with the reported travels of Homer, to allow an Eastern origin to a portion of his fable,” said Lathom. “After your banished Christians and roving monks,” said Herbert, “you would admit the Spanish Arabians.” “As one means, certainly,” replied Lathom; “and after them the Crusaders.” “It were almost superfluous,” rejoined Herbert, “to allude to the Crusades as further sources of romantic and didactic fiction. No one will dispute their right to a place in the system. About the period of the third crusade this kind of writing was at its height.” “Undoubtedly,” rejoined Lathom, “that age was the full tide of chivalry. Twenty years elapsed between that and the fourth and fifth expeditions into the east; and nearly a generation passed before, for the sixth and the last time, the wealth and blood of Europe was poured upon the plains of the East. Enough of money and life had been now spent to satisfy the most enthusiastic of the crusading body, and to check, if not to stem, the tide of popular feeling which had formerly run so strong in favor of the restoration of the sepulchre and the holy city to the guardianship of the faithful. Time was now at last beginning to allay the Anti-Saracenic passion. With the decline of these remarkable expeditions romantic fiction began to be regarded. For though originally extraneous and independent, romantic fictions had of late years become incorporated with chivalry and its institutions, and, with them, they naturally fell into decay.” “Come, come, we must break off this discussion,” said Thompson, “or else we shall have no time to judge of Lathom’s performance this evening.”
- 82. “The story I selected to begin with is one replete with eccentricity, and peculiarly characteristic of this age; it is entitled
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