T Cell Protocols Second Edition Angus Stock
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- 5. T Cell Protocols Second Edition Angus Stock Digital Instant Download Author(s): Angus Stock, Vincenzo Cerundolo (auth.), Gennaro De Libero (eds.) ISBN(s): 9781588295873, 1588295877 Edition: 2 File Details: PDF, 2.77 MB Year: 2009 Language: english
- 8. 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 TM John M. Walker, SERIES EDITOR 514. T Cell Protocols: Second Edition, edited by Gennaro De Libero, 2009 502. Bacteriophages: Methods and Protocols, Volume 2: Mole- cular and Applied Aspects, edited by Martha R. J. Clokie and Andrew M. Kropinski, 2009 501. Bacteriophages: Methods and Protocols, Volume 1: Isola- tion, Characterization, and Interactions, edited by Martha R. J. Clokie and Andrew M. Kropinski, 2009 496. DNA and RNA Profiling in Human Blood: Methods and Protocols, edited by Peter Bugert, 2009 493. Auditory and Vestibular Research: Methods and Proto- cols, edited by Bernd Sokolowski, 2009 490. Protein Structures, Stability, and Interactions, edited by John W. Schriver, 2009 489. Dynamic Brain Imaging: Methods and Protocols, edited by Fahmeed Hyder, 2009 485. HIV Protocols: Methods and Protocols, edited by Vinayaka R. Prasad and Ganjam V. Kalpana, 2009 484. Functional Proteomics: Methods and Protocols, edited by Julie D. Thompson, Christine Schaeffer-Reiss, and Marius Ueffing, 2008 483. Recombinant Proteins From Plants: Methods and Protocols, edited by Lóic Faye and Veronique Gomord, 2008 482. Stem Cells in Regenerative Medicine: Methods and Protocols, edited by Julie Audet and William L. Stanford, 2008 481. Hepatocyte Transplantation: Methods and Protocols, edited by Anil Dhawan and Robin D. Hughes, 2008 480. Macromolecular Drug Delivery: Methods and Proto- cols, edited by Mattias Belting, 2008 479. Plant Signal Transduction: Methods and Protocols, edi- ted by Thomas Pfannschmidt, 2008 478. Transgenic Wheat, Barley and Oats: Production and Characterization Protocols, edited by Huw D. Jones and Peter R. Shewry, 2008 477. Advanced Protocols in Oxidative Stress I, edited by Donald Armstrong, 2008 476. Redox-Mediated Signal Transduction: Methods and Protocols, edited by John T. Hancock, 2008 475. Cell Fusion: Overviews and Methods, edited by Eliza- beth H. Chen, 2008 474. Nanostructure Design: Methods and Protocols, edited by Ehud Gazit and Ruth Nussinov, 2008 473. Clinical Epidemiology: Practice and Methods, edited by Patrick Parfrey and Brendon Barrett, 2008 472. Cancer Epidemiology, Volume 2: Modifiable Factors, edited by Mukesh Verma, 2008 471. Cancer Epidemiology, Volume 1: Host Susceptibility Factors, edited by Mukesh Verma, 2008 470. Host-Pathogen Interactions: Methods and Protocols, edited by Steffen Rupp and Kai Sohn, 2008 469. Wnt Signaling, Volume 2: Pathway Models, edited by Elizabeth Vincan, 2008 468. Wnt Signaling, Volume 1: Pathway Methods and Mammalian Models, edited by Elizabeth Vincan, 2008 467. Angiogenesis Protocols: Second Edition, edited by Stewart Martin and Cliff Murray, 2008 466. Kidney Research: Experimental Protocols, edited by Tim D. Hewitson and Gavin J. Becker, 2008 465. Mycobacteria, Second Edition, edited by Tanya Par- ish and Amanda Claire Brown, 2008 464. The Nucleus, Volume 2: Physical Properties and Ima- ging Methods, edited by Ronald Hancock, 2008 463. The Nucleus, Volume 1: Nuclei and Subnuclear Com- ponents, edited by Ronald Hancock, 2008 462. Lipid Signaling Protocols, edited by Banafshe Lari- jani, Rudiger Woscholski, and Colin A. Rosser, 2008 461. Molecular Embryology: Methods and Protocols, Second Edition, edited by Paul Sharpe and Ivor Mason, 2008 460. Essential Concepts in Toxicogenomics, edited by Donna L. Mendrick and William B. Mattes, 2008 459. Prion Protein Protocols, edited by Andrew F. Hill, 2008 458. Artificial Neural Networks: Methods and Applications, edited by David S. Livingstone, 2008 457. Membrane Trafficking, edited by Ales Vancura, 2008 456. Adipose Tissue Protocols, Second Edition, edited by Kaiping Yang, 2008 455. Osteoporosis, edited by Jennifer J.Westendorf, 2008 454. SARS- and Other Coronaviruses: Laboratory Protocols, edited by Dave Cavanagh, 2008 453. Bioinformatics, Volume 2: Structure, Function, and Applications, edited by Jonathan M. Keith, 2008 452. Bioinformatics, Volume 1: Data, Sequence Analysis, and Evolution, edited by Jonathan M. Keith, 2008 451. Plant Virology Protocols: From Viral Sequence to Pro- tein Function, edited by Gary Foster, Elisabeth Johan- sen, Yiguo Hong, and Peter Nagy, 2008 450. Germline Stem Cells, edited by Steven X. Hou and Shree Ram Singh, 2008 449. Mesenchymal Stem Cells: Methods and Protocols, edi- ted by Darwin J. Prockop, Douglas G. Phinney, and Bruce A. Brunnell, 2008 448. Pharmacogenomics in Drug Discovery and Develop- ment, edited by Qing Yan, 2008 447. Alcohol: Methods and Protocols, edited by Laura E. Nagy, 2008 446. Post-translational Modifications of Proteins: Tools for Functional Proteomics, Second Edition, edited by Christoph Kannicht, 2008 445. Autophagosome and Phagosome, edited by Vojo Deretic, 2008 444. Prenatal Diagnosis, edited by Sinhue Hahn and Laird G. Jackson, 2008 443. Molecular Modeling of Proteins, edited by Andreas Kukol, 2008
- 9. 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 TM T Cell Protocols Second Edition Edited by Gennaro De Libero University of Basel, Basel, Switzerland
- 10. Editor Gennaro De Libero University of Basel Basel, Switzerland Gennaro.DeLibero@unibas.ch Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK ISSN: 1064-3745 e-ISSN: 1940-6029 ISBN: 978-1-58829-587-3 e-ISBN: 978-1-60327-527-9 DOI 10.1007/978-1-60327-527-9 Library of Congress Control Number: 2008941671 # Humana Press, a part of Springer ScienceþBusiness Media, LLC 2009 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper springer.com
- 11. Preface This book is a collection of protocols, to provide novel techniques for the study of the biology of T lymphocytes. The methods described in this book do not cover all of the techniques currently used to study T cell-mediated immune responses for the simple reason that T cell immunology is probably the immunological discipline which can be investigated with the widest variety of approaches. The choice of chapters was made taking into account two points: First, many of the techniques that have been used for some time have been upgraded during the past few years given the greater availability of a variety of products (i.e. cytokines, chemokines, monoclonal antibodies), of refined technical devices (i.e. novel cell culture and cell analysis equipments), and the development of novel instrumentation (i.e. multipara- metric flow cytometers, confocal microscopes). Therefore, in several chapters ‘‘old techniques’’, which remain fundamental to T cell immunology, are described in their ‘‘modern’’ versions. Secondly, the technical advancement has generated the possibility to establish novel assays to investigate T cell physiology. This is reflected in the chapters which describe the protocols that allow use of these modern approaches. The preparation of this book has required participation of several scientists, all leading experts in their respective fields. Without their enthusiastic participation, this work would not have been possible. Therefore, I thank all authors for their contribu- tions and accept all criticism for missing parts, or information or details, for which only I am responsible. I hope these protocols will be useful for young investigators who approach for the first time the complex field of immunology and for those more experienced scientists who look for concise and efficacious descriptions of novel methods. v
- 12. Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Color Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi 1 Analysis of Frequency and Phenotype of Antigen-Specific T Cells . . . . . . . . . . . . . . . . . 1 Angus Stock and Vincenzo Cerundolo 2 B Cell Helper Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Sergio Abrignani, Elena Tonti, Giulia Casorati, and Paolo Dellabona 3 transkingdom RNA Interference (tkRNAi): A Novel Method to Induce Therapeutic Gene Silencing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27 Thu A. Nguyen and Johannes H. Fruehauf 4 Flow Cytometry and Cell Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Sonia Gavasso 5 Investigating T Cells by Polychromatic Flow Cytometry . . . . . . . . . . . . . . . . . . . . . . . 47 Enrico Lugli, Leonarda Troiano, and Andrea Cossarizza 6 Generation of Human T Cell Clones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65 Sabrina Mariotti and Roberto Nisini 7 Limiting Dilution Analysis of Antigen-Specific T Cells . . . . . . . . . . . . . . . . . . . . . . . . 95 Jorge Carneiro, Lurdes Duarte, and Elisabetta Padovan 8 T Cell Epitope-Mapping by Cytokine Gene Expression Assay . . . . . . . . . . . . . . . . . . 107 Maurizio Provenzano and Giulio C. Spagnoli 9 Cytokine Multiplex Immunoassay: Methodology and (Clinical) Applications . . . . . . 119 Wilco de Jager, Berent Prakken, and Ger T. Rijkers 10 Purification of the T Cell Antigen Receptor and Analysis by Blue-Native PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135 Mahima Swamy and Wolfgang W.A. Schamel 11 Non-Replicating Recombinant Vaccinia Virus Expressing CD80 to Enhance T-Cell Stimulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151 Paul Zajac Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163 vii
- 13. Contributors SERGIO ABRIGNANI, MD Instituto Nazionale di Genetica Molecolare-INGM, Milan, Italy JORGE CARNEIRO, PHD Instituto Gulbenkian de Ciência, Oeiras, Portugal GIULIA CASORATI, PHD Experimental Immunology Unit, Cancer Immunotherapy and Gene Therapy Program, Department of Biology and Biotechnology, San Raffaele Scientific Institute, Milan, Italy VINCENZO CERUNDOLO, MD, PHD Nuffield Department of Clinical Medicine, Weatherall Institute of Molecular Medicine, Oxford, UK ANDREA COSSARIZZA, MD, PHD Department of Biomedical Sciences, Section of General Pathology, Modena, Italy WILCO DE JAGER, PHD Department of Pediatric Immunology, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Utrecht, The Netherlands GENNARO DE LIBERO, MD, PHD Experimental Immunology, Department of Research, University Hospital Basel, Basel, Switzerland PAOLO DELLABONA, MD, PHD Experimental Immunology Unit, Cancer Immunotherapy and Gene Therapy Program, Department of Biology and Biotechnology, San Raffaele Scientific Institute, Milan, Italy LURDES DUARTE, DIPL. BIOL. Instituto Gulbenkian de Ciência, Oeiras, Portugal JOHANNES H. FRUEHAUF, MD, PHD GI Cancer Laboratory, Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA SONIA GAVASSO Neurology Research Lab, Haukeland University Hospital, Gamle, Havedbgning, Bergen, Norway ENRICO LUGLI, BSC Department of Biomedical Sciences, Section of General Pathology, Modena, Italy SABRINA MARIOTTI, PHD Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Roma, Italy THU A. NGUYEN, BSC GI Cancer Laboratory, Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA ROBERTO NISINI, MD Dipartimento di Malattie Infettive, Parassitarie e Immunomediate, Istituto Superiore di Sanità, Roma, Italy ELISABETTA PADOVAN, PROF. PHD Universidade de Lisboa, Faculdade de Medicina, Lisboa, Portugal BERENT PRAKKEN, PROF. MD, PHD Department of Pediatric Immunology, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Utrecht, The Netherlands MAURIZIO PROVENZANO, MD Department of Urology, University Hospital of Zurich, Zurich, Switzerland ix
- 14. GER T. RIJKERS, PHD Department of Pediatric Immunology, Wilhelmina Children’s Hospital, University Medical Center Utrecht, Utrecht, The Netherlands; Laboratory of Medical Microbiology and Immunology, St. Antonius Hospital, Nieuwegein, The Netherlands WOLFGANG W.A. SCHAMEL, PHD Department of Molecular Immunology, Max Planck Institute for Immunobiology, University of Freiburg, Freiburg, Germany GIULIO C. SPAGNOLI, MD Institute of Surgical Research and Hospital Management, University Hospital Basel, Basel, Switzerland ANGUS STOCK, BSC Nuffield Department of Clinical Medicine, Weatherall Institute of Molecular Medicine, Oxford, UK MAHIMA SWAMY, MSC, ME Department of Molecular Immunology, Max Planck Institute for Immunobiology, University of Freiburg, Freiburg, Germany ELENA TONTI, PHD Experimental Immunology Unit, Cancer Immunotherapy and Gene Therapy Program, Department of Biology and Biotechnology, San Raffaele Scientific Institute, Milan, Italy LEONARDA TROIANO, BSC Department of Biomedical Sciences, Section of General Pathology, Modena, Italy PAUL ZAJAC, PHD University Hospital Basel, Institute of Surgical Research and Hospital Management, Basel, Switzerland x Contributors
- 15. Color Plates Color Plate 1: Multidimensional analysis of human PBMC stimulated with either IL-6 (green), IL-4 (red) or left untreated (blue). Cells were fixed and permeabilized following protocol 3.2 and stained simultaneously with antibody cocktail A. Top panels show superim- posed dot plots and histograms for T-cells (CD3+ ), the bottom panels show B-cells (CD20+ ). In overlays the induction of specific phosphor- ylation events are clearly identifiable. (see discussion on p. 41) Color Plate 2: PBMC were stimulated with indicated cytokines, fixed and permea- bilized according to protocol 3.2. T-cells (CD3+ ) and B-cells (CD20+ ) were gated according to markers while monocytes were gated in scatter plot. Open histograms represent untreated cells, filled histograms stimulated cells. Induction of phos- phorylation is clearly identifiable (filled yellow histograms). (see discus- sion on p. 41) Color Plate 3: Visualization of the data generated by the FACS analysis following protocol 3.2. The columns represent the cell subsets, T-cells, B-cells, monocytes. Each row represents a cytokine stimulation stained with one of the antibody cocktails and subsequently analyzed for the indicated phos- phoprotein. The color of each block represents the fold change (log2) in MFI in the channel corresponding to the analyzed phophorylated protein. (see discussion on p. 42) xi
- 16. Chapter 1 Analysis of Frequency and Phenotype of Antigen-Specific T Cells Angus Stock and Vincenzo Cerundolo Abstract Over the last decade, our understanding of the cellular immune system has been greatly advanced through the development of methods to identify antigen-specific T cells directly ex vivo. The major reagents and techniques used for this purpose are (i) tetramerised MHC:peptide complexes (tetramers) which bind to specific T-cell receptors (TCR) and (ii) assays that detect T cells which synthesise cytokines in response to cognate stimulation (intracellular cytokine staining (ICS)). Here, we provide a detailed description of the procedure for generating and using class I MHC:peptide tetramers to label peptide-specific T cells and for carrying out ICS to measure antigen-specific T lymphocytes. Key words: Tetramers, antigen-specific T cells, MHC class I, intracellular cytokine staining, CTL. 1. Introduction Accurate measurements of MHC class I restricted T cells have been hampered by the lack of staining reagents capable of identi- fying antigen-specific cytotoxic T lymphocytes (CTL). Until a few years ago, assays used to detect CTL depended on in vitro culture of antigen-specific CTL and relied on the ability of expanded CTL either to kill target cells or to secrete relatively large amounts of lymphokines. Limiting dilution assay (LDA) was used to quantify CTL precursor frequency and Cr51 release assay was used to assess CTL specificity. Both assays were unable to detect cells incapable to proliferate or to show cytotoxic effector function. New meth- ods for detection of antigen-specific CTL have recently been developed (i.e. ELISPOT and intracellular cytokine staining (ICS)) and have allowed to measure in ex vivo assays the frequency Gennaro De Libero (ed.), T Cell Protocols: Second Edition, vol. 514 Ó 2009 Humana Press, a part of Springer ScienceþBusiness Media DOI 10.1007/978-1-60327-527-9_1 Springerprotocols.com 1
- 17. of cytokine-secreting effector cells (1–4). Although these assays can reliably measure the frequency of specific CTL able to secrete cytokines, they fail to detect resting or naı̈ve CTL, unable to secrete lymphokines in ex vivo assays. Over the last few years, a novel method has been developed based on the generation of fluorogenic tetrameric HLA class I molecules loaded with defined peptide epitopes, which is independent of the ability of cells to proliferate and secrete lymphokines (5). The use of HLA class I tetramers has allowed the characterisation and monitoring of specific viral and tumour responses and has provided an opportu- nity to greatly accelerate the development of new vaccination strategies (reviewed in (6)). Here we will describe Materials and Methods to engineer MHC class I tetramers and to carry out combined MHC class I tetramer and ICS of antigen-specific T lymphocytes. 2. Materials 2.1. MHC Class I: Peptide Tetramers 1. pET expression systems (Novagen) 2. Oligonucleotide primers, DNA polymerase, ligase and restriction enzymes 3. Agarose and DNA-sequencing systems 4. Ampicillin (used at 100 mg/ml unless otherwise stated) 5. IPTG 6. Triton wash: 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 0.5% Triton, 10 mM DTT, 1 mM EDTA and 0.1% azide 7. Re-suspension buffer: 50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 10 mM DTT and 1 mM EDTA 8. Urea solution: 8 M urea, 10 mM Tris–HCl (pH 8.0), 100 mM NaH2PO4, 0.1 mM EDTA and 10 mM DTT 9. BCA protein assay kit 10. Reagents and instruments for SDS gel 11. 0.45 mM filter membrane 12. Refolding buffer: 100 mM Tris–HCl (pH 8.0), 400 mM l- arginine hydrochloride, 2 mM EDTA, 5 mM reduced glu- tathione, 0.5 mM oxidised glutathione and 0.1 mM PMSF (diluted in H2O) 13. Minimal peptide 14. Amicon stir cell (Diaflo PM10 150 mm membrane) 15. FPLC 2 Stock and Cerundolo
- 18. 16. Tris-buffered saline 17. MgCl2(stock solution, 50 mM) 18. Leupeptin (stock solution, 10 mM) 19. Pepstatin (stock solution, 10 mM) 20. d-Biotin (stock solution, 4 mM) 21. ATP (stock solution, 50 mM) 22. TBS 23. Bir A Enzyme (stock solution, 500 mM) 24. Phycoerythrin-conjugated streptavidin 25. ELISA reagents 26. Streptavidin-peroxidase 27. FACS buffer: PBS containing 1% BSA and 0.02% sodium azide 28. Antibodies against T-cell co-receptors (CD8 and CD4) 29. Propidium iodide 30. Formaldehyde 2.2. Intracellular Cytokine Staining 1. Synthetic peptides 2. Brefaldin A (diluted in methanol) 3. Antibodies against surface antigens (e.g. CD8, CD4, etc.) and intracellular cytokines (e.g. IFN-, IL-2, TNF-, IL-4, IL-10, etc.) 4. Formaldehyde: 1% diluted in PBS with 0.1% sodium azide 5. Saponin 6. T-cell medium: RPMI 1640 supplemented with 10% heat- inactivated foetal calf serum and 5% supplementum comple- mentum (SC: HEPES 23.83 g/l, benzylpenicillin 2 106 U/l, streptomycin 2 g/l, 1-glutamine 6 g/l for RPMI, 50 mM 2- mercaptoethanol) 7. FACS buffer: PBS containing 1% BSA and 0.02% sodium azide. 3. Methods Here we shall provide protocols for the two most commonly used flow cytometry-based techniques for enumerating antigen-speci- fic T cells: (1) MHC tetramer analysis and (2) ICS. We shall describe the procedure for generating of class I MHC:peptide tetramers (Section 3.1) and using these reagents to stain Frequency Analysis of Antigen-Specific T Cells 3
- 19. population of cells for identifying antigen-specific T cells (Section 3.2). Finally, the protocol for using ICS to identify both peptide- and virus-specific T cells will be described (Section 3.3). 3.1. Production of MHC Class I:Peptide Tetrameric Complexes The strategy for generating class I MHC:peptide complexes was first described by Garboczi and Wiley in 1992 (7). In this method, the 2-microglobulin (2-m) and the MHC heavy chains are expressed separately in prokaryotic expression systems. These molecules are then purified from bacterial inclusion bodies and refolded in the presence of peptide. However, monomeric com- plexes bind TCR poorly, necessitating the formation into multi- mers to allow cooperative TCR binding. The method for combining MHC:peptide monomers into tetrameric complexes for staining antigen-specific T cells was devised by Altman and Davis in 1996 (5). In this protocol, refolded monomers (which are fused to the Bir A substrate peptide) are biotinylated with the BirA enzyme and tetramerised with fluorochrome-conjugated streptavidin. Here, we shall provide an outline for this procedure, although it should be noted that customised tetramers are com- mercially available and may be purchased from outside sources. 3.1.1. Expression of MHC Chains The most commonly used vectors to express MHC proteins are the pET plasmids (although other vectors may be used in their place). Genes inserted into the multiple cloning site of the pET plasmids are expressed as a T7 gene product, induced through the addition of IPTG. 1. Amplify the extracellular domains of the MHC class I heavy chains (1, 2, and 3) or 2-m from a cDNA or/and existing plasmid and ligate into separate expression vectors. In the case of the MHC heavy chain, insert sequence immediately upstream of the BirA substrate peptide (BSP) sequence to produce MHC–BSP fusion product. 2. Transform plasmids into E. coli and incubate over night at 37°C with ampicillin selection (100 mg/ml). Pick single colo- nies, reselect with ampicillin and screen for the desired plasmid through enzyme digestion and sequence analysis. 3.1.2. Induction of MHC Protein 1. Using a single colony containing either the heavy chain or 2- m expression vectors, inoculate 10 ml LB (+ampicillin selec- tion) and grow overnight (37°C, shaking). Use 1 ml of this ‘starter’ broth to inoculate ‘bulk’ 200 ml LB (+ampicillin selection) and culture overnight (37°C, shaking). 2. The following day, add 50 ml of the bulk culture to each of four flasks containing 1 l LB (+ampicillin selection). Culture at 37°C with shaking, until the OD600 reaches 0.6 (this takes 3–4 h). Take a 1.5 ml sample to analyse pre-induction pro- tein expression (see Fig. 1.1). 4 Stock and Cerundolo
- 20. 3. Induce the expression of MHC proteins by adding IPTG (final concentration 0.5 mM) and incubate for 4 h (37°C, shaking) to allow MHC protein expression. 4. Take a 1.5 ml post-induction sample for gel analysis (see Fig. 1.1), measuring the OD600 to equilibrate total pre- and post-induction bacterial concentration. 5. Pellet the remaining culture (20 min, 5000 g, 4°C). Decant supernatant and resuspend in ice-cold PBS (10 ml per flask). Here, bacteria may be frozen at –80°C and stored overnight before inclusion body purification, or purified directly. 3.1.3. Inclusion Body Isolation 1. If frozen, thaw pellet on ice. Lyse bacteria by sonicating in bursts of 30 s, keeping on ice between bursts to prevent over- heating. Repeat until suspension reaches milk-like consistency (this normally requires 4 rounds of sonication). 2. Transfer suspension to centrifuge tubes and spin for 10 min (13,000 g, 4°C). Both inclusion bodies (which should appear white) and cell debris (which appear as darkish matter) should pellet. If the supernatant remains cloudy re-spin. 3. To remove cell debris, resuspend cell pellet in 25 ml of cold triton wash buffer. Spin for 10 min (13,000 g, 4°C) and discard supernatant. Wash another 2–3 times in triton buffer or until the supernatant has become clear (indicating that bacterial debris has been removed). Fig. 1.1. Purification of MHC proteins. Protein extracts from bacteria transformed with 2-m expression vectors taken prior to IPTG induction (pre-induction), 4 h after IPTG addition (post-induction) and following urea purification were resolved on a 15% SDS-PAGE gel and stained with Coomassie blue. Note the induction and purification of the 2-m sized band. Frequency Analysis of Antigen-Specific T Cells 5
- 21. 4. To remove detergent from inclusion body sample, resuspend the pellet in 25 ml of cold re-suspension buffer and pellet (10 min, 13,000 g, 4°C). 5. Dissolve inclusion bodies by resuspending in a total volume of 25 ml fresh urea solution, mixing with a glass homogeniser. Rotate overnight at 4°C. 6. Spin urea extracts for 10 min (13,000 g, 4°C). Working on ice, pool supernatant and take a sample for gel analysis (see Fig. 1.1). 7. Determine protein concentration (using BCA analysis) and aliquot into 13 mg portions. Store at –80°C for up to 1 month before refolding. 8. For gel analysis, pellet 1 ml of pre-induction and post-induc- tion samples and resuspend in double-distilled water at a volume of 100 ml OD600 (equilibrating protein concentra- tion). Run 10 ml of these resuspended samples alongside the urea-purified protein on a SDS gel (10% gel for MHC heavy chains and 15% for 2-m: see Fig. 1.1). 3.1.4. Refolding and Purification of Class I MHC:Peptide Monomers The MHC heavy chain and 2-m proteins are mixed with the target peptide, refolding into monomeric MHC:peptide com- plexes. Monomers are then purified by chromatography. 1. Make up 1 l of refolding buffer and filter through a 0.45 mm membrane. Collect into a 2 l conical flask and cool to 4°C. 2. Dissolve 10 mg of peptide in 1 ml DMSO immediately before refolding. 3. Stir refolding buffer at 4°C and add dissolved peptide (final concentration 10 mM), 26 mg 2-m protein (final concen- tration 2 mM) and 32 mg MHC heavy chain protein (final concentration 1 mM). Refold for 40 h at 4°C with stirring. 4. To concentrate the refolded protein, filter the mix through a 0.45 mM membrane to remove precipitated proteins and load into an Amicon stir cell with a Diaflo PM10 150 mm mem- brane. Concentrate to 7 ml volume. 5. Refilter concentrate through a 0.45 mM membrane and purify the monomer using FPLC with Tris-buffered saline as the running buffer. Collect fractions of appropriate molecular weight and concentrate to below 3 ml using an Amicon stir cell (working at 4°C to limit protein degradation). 3.1.5. Biotinylation of Class I MHC:Peptide Monomers At this stage, monomeric class I MHC:peptides are biotinylated through the addition of the BirA enzyme. This enzyme biotiny- lates the BSP that is fused to the MHC heavy chain. 1. Transfer the purified monomers to a 50 ml Falcon tube and add – MgCl2 (final concentration 5 mM) 6 Stock and Cerundolo
- 22. – Leupeptin (final concentration 1 mM) – Pepstatin (final concentration 1 mM) – d-Biotin (final concentration 400 mM) – ATP (dissolved in TBS: final concentration 5 mM) – Bir A enzyme (final concentration 50 mM) Make up to a final volume of 4 ml with TBS, mix through inversion and incubate overnight at room temperature. 2. Filter the biotinylated complex through a 0.45 mm mem- brane and purify with FPLC (Tris-buffered saline running buffer). Collect fractions of appropriate molecular weight and immediately add leupeptin and pepstatin (final con- centration of each at 1 mM) to prevent protein degradation. 3. Concentrate biotinylated complexes on an Amicon cell to a final volume of around 1 ml. 4. Determine protein concentration (i.e. with a BCA assay) and aliquot into 100 mg portions. Quick-freeze monomers in liquid nitrogen and store at –80°C until tetramerisation. 5. See Notes for optional step (see Note 1). 3.1.6. Tetramerisation of Class I MHC:Peptide Monomer Monomers are tetramerised through the addition of fluoro- chrome-conjugated streptavidin. To achieve maximal tetramer- isation, streptavidin is initially added to an excess of monomer. Limiting avidin ensures that all biotin-binding sites are occu- pied with MHC monomers, promoting the formation of tetra- meric complexes. The concentration of avidin is then progressively increased until all MHC has gone into tetramers and the avidin is saturating. Here, ExtrAvidin–Phycoerythrin (Sigma: E-4011) is used at a final concentration of 1 ml/mg of monomer (for instance, a total of 200 ml of avidin–PE to tetramerise 200 mg monomer). 1. Initially add half the total avidin–PE volume to monomer, mixing gently with a pipette tip. Incubate on ice for 30 min (keep in dark to protect fluorochrome). 2. Divide the remaining avidin–PE into five equal volumes. 3. Add one aliquot of avidin–PE (equating to one tenth of total volume) to monomer. Mix and incubate for 20 min on ice (dark). 4. Repeat step 3 another four times until the entire volume of avidin–PE has been added. 5. Test biotin availability following tetramerisation by ELISA. Plate out equivalent concentrations of monomer and tetramer in ELISA plates and perform twofold serial dilution (across eight wells). Perform standard ELISA, probing for biotin using Frequency Analysis of Antigen-Specific T Cells 7
- 23. streptavidin-peroxidase. Upon developing, the tetramer sam- ple should appear to contain 1/8–1/16 as much biotin as the monomer. If biotin levels are in excess of this, repeat steps 3 and 4 until avidin has saturated biotin sites. 3.2. Using Class I MHC:Peptide Tetrameric Complexes for Frequency Analysis of Antigen-Specific T Cells In this section, we shall describe the staining protocol for using class I MHC:peptide tetramers to identify antigen-specific T cells in whole lymphocyte populations. 3.2.1. Staining Protocol for Class I MHC Tetramers 1. Prepare single lymphocyte suspension from blood or organ tissues of test and control samples (see Note 2). 2. Lyse red blood cells and count live lymphocytes. 3. Transfer 1–2 106 live lymphocytes into staining vessels (usually 5 ml FACS tubes or 96-well plates). Pellet cells by centrifugation (5 min, 500 g) and wash twice with FACS buffer. 4. Stain cells first with tetramer by resuspending the cell pellet in 50 ml tetramer complex diluted to the optimal working con- centration in FACS buffer (see Note 3). Incubate for 30 min at 37°C in the dark (see Note 4). 5. Wash cells with FACS buffer and pellet (5 min, 500 g). 6. Next stain for T-cell co-receptors. Resuspend cell pellet in a saturating concentration of antibodies (50 ml volume diluted in FACS buffer) against CD8 (for class II tetramers stain for CD4). In addition, antibodies directed against T-cell surface antigens may be included at this step (see Note 5). Incubate for 30 min on ice in the dark. 7. Wash cells twice in FACS buffer and prepare for flow cyto- metric analysis. The sample may be analysed directly as live cells or fixed and stored for up to 2 days before analysis. a For live analysis, resuspend cell pellet in 200 ml FACS buffer. Just prior to flow cytometry (between 1 and 30 min) add propidium iodide (final concentration at 2.5–5 mg/ml), to allow dead cell exclusion during analysis (see Note 6). b For fixation, resuspend cell pellet in 100 ml of 1% formalde- hyde and incubate for 20 min at room temperature (dark). Following fixation, wash cells twice in PBS. Resuspend in 200 ml PBS and store at 4°C in the dark until flow cytometry. 8 Stock and Cerundolo
- 24. 3.2.2. Flow Cytometric Analysis of Tetramer Staining 1. For analysis, set up FSC versus SSC plot and set the primary gate (R1) upon lymphocyte-sized cells. 2. Where propidium iodide (PI) has been included set the sec- ondary gate (R2) upon PI-negative lymphocytes. Combine gates (R1 R2) to restrict analysis to live lymphocytes (see Fig. 1.2). 3. To analyse the frequency of antigen-specific T cells, show tetramer staining versus CD8 expression. Traditionally, the frequency of antigen-specific T cells is expressed as the percentage of T cells (CD4 or CD8) that stain positive with the MHC:peptide tetramer complex. Alternatively, Fig. 1.2. Quantitation of antigen-specific T cells by tetramer staining. Blood from naı̈ve C57BL/6 mice and mice infected 7 days earlier with herpes simplex virus (HSV) were stained with an APC-conjugated anti-CD8 antibody and the PE-conjugated tetramer of the dominant HSV peptide in complex with H2-Kb . Frequency analysis was restricted to live lymphocytes (R1 R2) by gating upon lymphocyte-sized events (R1) that were PI negative (R2). Inset values show the percentage of CD8+ T cells that are tetramer positive for naı̈ve and infected samples. Note that the HSV tetramer-specific population represents less than 0.1% of CD8+ T cells from naı̈ve mice before expanding to around 9% of circulating CD8+ T cells after HSV infection. Frequency Analysis of Antigen-Specific T Cells 9
- 25. when cells are from organ samples, the total number of antigen-specific T cells per organ may be a more appropriate value (see Fig. 1.2). 3.3. Analysis of Antigen-Specific T Cells Using Intracellular Cytokine Staining Upon recognition of their cognate antigen, T cells synthesise and secrete a range of cytokines, including IFN-, TNF-, IL-2, IL-4 and IL-10. The ICS assay utilises this activity, identifying antigen-specific T cells through their production of cytokines in response to antigen. In this assay, the secretory pathway is blocked, causing the intracellular accumulation of nascent cyto- kines, which in turn are detected by antibody staining and flow cytometry (1–4). The primary advantage of the ICS versus tetramer staining is that precise knowledge of T-cell epitopes is not required. Specifi- cally, the T-cell response against an entire pathogen may be enumerated following activation with stimulator cells that are infected with particular pathogen. However, peptide-specific responses are also routinely measured following stimulation with minimal peptides. 3.3.1. Preparation of Virus-Infected Stimulator Cells To induce cytokine synthesis, responder T cells can be stimu- lated with either minimal peptides (stimulating peptide-specific T cells) or with virus-infected stimulator cells (stimulating virus-specific T cells). A number of cell types have been used as virus-infected stimulator cells, including fibroblasts (8) and dendritic cells (9). Additionally, the lymphocyte sample may be directly infected (10). The following section describes a generalised method for generating such infected stimulator cells. 1. Wash stimulator cells with serum-free media, aspirate super- natant and resuspend in a minimal volume of serum-free media containing the target virus at a multiplicity of infec- tion (m.o.i) of between 1 and 5. Incubate cells for 60 min at 37°C. 2. Aspirate media and replace with an excess volume of growth media (e.g. RPMI with 10% FCS). Incubate cells for 4–16 h at 37°C to allow virus protein synthesis (see Note 7). 3. Following incubation, aspirate infectious supernatant, harvest cells and resuspend in T-cell media for use as virus-infected stimulators. 3.3.2. In Vitro Stimulation of T Cells 1. Prepare single lymphocyte cell suspensions from blood or organ tissue from test and control samples. Lyse red blood cells and wash in T-cell growth media (see Note 2). 10 Stock and Cerundolo
- 26. Discovering Diverse Content Through Random Scribd Documents
- 27. Fig. 23. Details of Bleriot Monoplane Motor. The motor regularly employed is the 30-horse-power, three-cylinder Anzani, a two-cylinder type of which is shown in Aeronautical Motors Fig. 40. From the amateur's standpoint, a disadvantage of the Bleriot is the very short space allowed for the installation of the motor. For this reason, the power plant must be fan shaped, like the Anzani; star form, like the Gnome; or of the two-cylinder opposed type. It must likewise be air-cooled, as there is no space available for a radiator. Fig. 24. Side Elevation of Bleriot Monoplane
- 28. Fig. 25. Top and Side View of Bleriot Fuselage on Which Machine Is Assembled Fuselage. Like most monoplanes, the Bleriot has a long central body, usually termed fuselage, to which the wings, running gear, and controls are all attached. A drawing of the fuselage with all dimensions is reproduced in Fig. 25, and as the machine is, to a large extent, built up around this essential, its construction is taken up first. It consists of four long beams united by 35 crosspieces. The beams are of ash, 1 3/16 inches square for the first third of their length and tapering to 7/8 inch square at the rear ends. Owing to the difficulty of securing good pieces of wood the full length, and also to facilitate packing for shipment, the beams are made in halves, the abutting ends being joined by sleeves of 1 1/8-inch, 20- gauge steel tubing, each held on by two 1/8-inch bolts. Although the length of the fuselage is 21 feet 11 1/4 inches, the beams must be made of two 11-foot halves to allow for the curve at the rear ends.
- 29. Fig. 26. Details of U-bolt Which is a Feature of Bleriot Construction The struts are also of ash, the majority of them being 7/8 by 1 1/4 inches, and oval in section except for an inch and a half at each end. But the first, second, and third struts (counting from the forward end) on each side, the first and second on the top, and the first strut on the bottom are 1 3/16 inches square, of the same stock as the main beams. Practically all of the struts are joined to the main beams by U-bolts, as shown by the detail drawing, Fig. 26, this being one of Louis Bleriot's inventions. The small struts are held by 1/8-inch bolts and the larger ones by 3/16-inch bolts. The ends of the struts must be slotted for these bolts, this being done by drilling three holes in a row with a 5/32- or 7/32-inch drill, according to whether the slot is for the smaller or larger size bolt. The wood between the holes is cut out with a sharp knife and the slot finished with a coarse, flat file. All of the U-bolts measure 2 inches between the ends. The vertical struts are set 1 inch forward of the corresponding horizontal
- 30. struts, so that the four holes through the beam at each joint are spaced 1 inch apart, alternately horizontal and vertical. To the projecting angles of the U-bolts are attached the diagonal truss wires, which cross all the rectangles of the fuselage, except that in which the driver sits. This trussing should be of 20-gauge piano wire (music-wire gauge) or 1/10-inch cable, except in the rectangles bounded by the large struts, where it should be 25-gauge piano wire or 3/32-inch cable. Each wire, of course, should have a turnbuckle. About 100 of these will be required, either of the spoke type or the regular type, with two screw eyes—the latter preferred. Transverse squares, formed by the two horizontal and two vertical struts at each point, are also trussed with diagonal wires. Although turnbuckles are sometimes omitted on these wires, it takes considerable skill to get accurate adjustments without them. The extreme rear strut to which the rudder is attached, is not fastened in the usual way. It should be cut with tongues at top and bottom, fitting into notches in the ends of the beams, and the whole bound with straps of 20-gauge sheet steel, bolted through the beams with 1/8-inch bolts. Continuing forward, the struts have no peculiarity until the upper horizontal one is reached, just behind the driver's seat. As it is impossible to truss the quadrangle forward of this strut, owing to the position of the driver's body, the strut is braced with a U-shaped half-round strip of 1/2 by 1 inch of ash or hickory bolted to the beams at the sides and to the strut at the rear, with two 1/8-inch bolts at each point. The front side of the strut should be left square where this brace is in contact with it. The brace should be steam
- 31. bent with the curves on a 9-inch radius, and the half-round side on the inside of the curve. The vertical struts just forward of the driver's seat carry the inner ends of the rear wing beams. Each beam is attached with a single bolt, giving the necessary freedom to rock up and down in warping the wings. The upper 6 inches of each of these struts fits into a socket designed to reinforce it. In the genuine Bleriot, this socket is an aluminum casting. However, a socket which many would regard as even better can be made from a 7-inch length of 20-gauge 1 1/8-inch square tubing. One end of the tube is sawed one inch through the corners; two opposite sides are then bent down at right angles to form flanges, and the other two sides sawed off. A 1- by 3- inch strip of 20-gauge sheet steel, brazed across the top and flanges completes the socket. With a little care, a very creditable socket can be made in this way. Finally, with the strut in place, a 3/8-inch hole is drilled through 4 inches from the top of the socket for the bolt securing the wing beam. The upper horizontal strut at this point should be arched about six inches to give plenty of elbow room over the steering wheel. The bending should be done in a steam press. The strut should be 1 3/16 inches square, cut sufficiently long to allow for the curve, and fitted at the ends with sockets as described above, but set at an angle by sawing the square tube down further on one side than on the other. On the two lower beams, is laid a floor of half-inch boards, extending one foot forward and one foot back of the center line of the horizontal strut. This floor may be of spruce, if it is desired to save a little weight, or of ordinary tongue-and-grooved floor boards,
- 32. fastened to the beams with wood screws or bolts. The horizontal strut under this floor may be omitted, but its presence adds but little weight and completes the trussing. Across the top of the fuselage above the first upper horizontal strut, lies a steel tube which forms the sockets for the inner end of the front wing beams. This tube is 1 3/4 inches diameter, 18 gauge, and 26 3/4 inches long. It is held fast by two steel straps, 16 gauge and 1 inch wide, clamped down by the nuts of the vertical strut U-bolts. The center of the tube is, therefore, in line with the center of the vertical struts, not the horizontal ones. The U-bolts which make this attachment are, of course, the 3/16-inch size, and one inch longer on each end than usual. To make a neat job, the tube may be seated in wood blocks, suitably shaped, but these must not raise it more than a small fraction of an inch above the top of the fuselage, as this would increase the angle of incidence of the wings. The first vertical struts on each side are extras, without corresponding horizontal ones; they serve only to support the engine. When the Gnome motor is used, its central shaft is carried at the centers of two X-shaped, pressed-steel frames, one on the front side, flush with the end of the fuselage and one on the rear. Truss Frame Built on Fuselage. In connection with the fuselage may be considered the overhead truss frame and the warping frame. The former consists of two inverted V's of 20-gauge, 1- by 3/8-inch oval tubing, joined at their apexes by a 20-gauge, 3/4-inch tube. Each V is formed of a single piece of the oval tubing about 5 feet long. The flattened ends of the horizontal tube are fastened by a bolt in the angles of the V's. The center of the horizontal tube should be 2 feet above the top of the fuselage. The
- 33. flattened lower ends of the rear V should be riveted and brazed to strips of 18-gauge steel, which will fit over the bolts attaching the vertical fuselage struts at this point. The legs of the front V should be slightly shorter, as they rest on top of the wing socket tube. Each should be held down by a single 3/16-inch bolt, passing through the upper wall of the tube and its retaining strap; these bolts also serve the purpose of preventing the tube from sliding out from under the strap. Each side of the frame is now braced by diagonal wires (No. 20 piano wire, or 1/14-inch cable) with turnbuckles. At the upper corners of this frame are attached the wires which truss the upper sides of the wings. The front wires are simply fastened under the head and nut of the bolt which holds the frame together at this corner. The attachment of the rear wires, however, is more complex, as these wires must run over pulleys to allow for the rocking of the rear wing beams when the wings are warped. To provide a suitable place for the pulleys, the angle of the rear V is enclosed by two plates of 20-gauge sheet steel, one on the front and one on the rear, forming a triangular box 1 inch thick fore and aft, and about 2 inches on each side, only the bottom side being open. These plates are clamped together by a 3/16-inch steel bolt, on which are mounted the pulleys. There should be sufficient clearance for pulleys 1 inch in diameter. The wires running over these pulleys must then pass through holes drilled in the tube. The holes should not be drilled until the wings are on, when the proper angle for them can be seen. The cutting and bending of the steel plates is a matter of some difficulty, and should not be done until the frame is otherwise assembled, so that paper patterns can be cut for them. They should have flanges bent around the tube, secured by
- 34. the bolts which hold the frame together, to keep them from slipping off. The oval tubing is used in the vertical parts of this frame, principally to reduce the wind resistance, being placed with the narrow side to the front. However, if this tubing be difficult to obtain, or if price is a consideration, no harm will be done by using 3/4-inch round tubing. Beneath the floor of the driver's cockpit in the fuselage is the warping frame, the support for the wires which truss the rear wing beams and also control the warping. This frame is built up of four 3/4-inch, 20-gauge steel tubes, each about 3 feet long, forming an inverted, 4-sided pyramid. The front and back pairs of tubes are fastened to the lower fuselage beams with 3/16-inch bolts at points 15 inches front and back of the horizontal strut. At their lower ends the tubes are joined by a fixture which carries the pulleys for the warping wires and the lever by which the pulleys are turned. In the genuine Bleriot, this fixture is a special casting. However, a very neat connection can be made with a piece of 1/16-inch steel stock, 1 1/4 by 6 inches, bent into a U- shape with the legs 1 inch apart inside. The flattened ends of the tubes are riveted and brazed to the outside upper corners of the U, and a bolt to carry the pulleys passes through the lower part, high enough to give clearance for 2-inch pulleys. This frame needs no diagonal wires. Running Gear. Passing now to the running gear, the builder will encounter the most difficult part of the entire machine, and it is impossible to avoid the use of a few special castings. The general plan of the running gear is shown in the drawing of the complete machine. Figs. 23 and 24, while some of the details are illustrated in
- 35. Fig. 27, and the remainder are given in the detail sheet, Fig. 28. It will be seen that each of the two wheels is carried in a double fork, the lower fork acting simply as a radius rod, while the upper fork is attached to a slide which is free to move up and down on a 2-inch steel tube. This slide is held down by two tension springs, consisting of either rubber tubes or steel coil springs, which absorb the shocks of landing. The whole construction is such that the wheels are free to pivot sideways around the tubes, so that when landing in a quartering wind the wheels automatically adjust themselves to the direction of the machine. A FRENCH DEVELOPMENT OF THE WRIGHT MACHINE BUILT UNDER THE WRIGHT PATENTS
- 36. There is Little Resemblance to the Original Except in Wing Form and Warping Framework. The main framework of the running gear consists of two horizontal beams, two vertical struts, and two vertical tubes. The beams are of ash, 4 3/4 inches wide in the middle half, tapering to 3 3/4 inches at the ends, and 5 feet 2 3/4 inches long overall. The upper beam is H inch thick and the lower 1 inch. The edges of the beams are rounded off except at the points where they are drilled for bolt holes for the attachment of other parts. The two upper beams of the fuselage rest on these beams and are secured to them by two 3/16-inch bolts each. The vertical struts are also of ash, 1 3/16 inch by 3 inches and 4 feet 2 inches long overall. They have tenons at each end which fit into corresponding square holes in the horizontal beams. The two lower fuselage beams are fastened to these struts by two 3/16-inch through bolts and steel angle plates formed from 1/16-inch sheet steel. The channel section member across the front sides of these struts is for the attachment of the motor, and will be taken up later. The general arrangement at this point depends largely on what motor is to be used, and the struts should not be rounded or drilled for bolt holes until this has been decided. From the lower ends of these struts CC, Fig. 27, diagonal struts DD run back to the fuselage. These are of ash, 1 3/16 by 2 1/2 inches and 2 feet inches long. The rear ends of the struts DD are fastened to the fuselage beams by the projecting ends of the U-bolts of the horizontal fuselage struts, and also by angle plates of sheet
- 37. steel. At the lower front ends the struts DD are fastened to the struts CC and the beam E by steel angle plates, and the beam is reinforced by other plates on its under side. Trussing. In the genuine Bleriot, the framework is trussed by a single length of steel tape, 1 1/8 by 1/16 inch and about 11 feet long, fastened to U-bolts in the beam A, Fig. 27. This tape runs down one side, under the beam E, and up the other side, passing through the beam in two places, where suitable slots must be cut. The tape is not made in this country, but must be imported at considerable expense. Ordinary sheet steel will not do. If the tape can not be obtained, a good substitute is 1/8-inch cable, which then would be made in two pieces and fastened to eye bolts at each end. Fig. 27. Details of Bleriot Running Gear
- 38. Fig. 28. Details of Various Fittings for Bleriot Monoplane The two steel tubes are 2 inches in diameter, 18-gauge, and about 4 feet 10 inches long. At their lower ends they are flattened, but cut away so that a 2-inch ring will pass over them. To these flattened ends are attached springs and wires which run from each tube across to the hub of the opposite wheel. The purpose of these is simply to keep the wheels normally in position behind the tubes. The tubes, it will be noticed, pass through the lower beam, but are sunk only 1/8 inch into the upper beam. They are held in place by sheet-steel sockets on the lower side of the upper beam and the upper side of the lower beam. The other sides of the beams are provided with flat plates of sheet steel. The genuine Bleriot has these sockets stamped out of sheet steel, but as the amateur builder will not have the facilities for doing this, an alternative construction is given here.
- 39. In this method, the plates are cut out to pattern, the material being sheet steel 1/16 inch thick, and a 1/2-inch hole drilled through the center, a 2-inch circle then being drawn around this. Then, with a cold chisel a half dozen radial cuts are made between the hole and the circle. Finally this part of the plate is heated with a blow-torch and a 2-inch piece of pipe driven through, bending up the triangular corners. These bent up corners are then brazed to the tubes, and a strip of light sheet steel is brazed on to cover up the sharp edges. Of course, the brazing should not be done until the slides GG, Figs. 27 and 28, have been put on. When these are once in place, they have to stay on and a breakage of one of them, means the replacement of the tube as well. This is a fault of the Bleriot design that can not well be avoided. It should be noticed that the socket at the upper end, as well as its corresponding plate on the other side of the beam, has extensions which reinforce the beam where the eye bolts or U-bolts for the attachment of the steel tape pass through. Forks. Next in order are the forks which carry the wheels. The short forks JJ, Figs. 27 and 28, which act simply as radius rods, are made of 1- by 3/8-inch oval tubing, a stock size which was specified for the overhead truss frame. It will be noticed that these are in two parts, fastened together with a bolt at the front end. The regular Bleriot construction calls for forged steel eyes to go in the ends of tubes, but these will be hard to obtain. The construction shown in the drawings is much simpler. The ends of the tubes are heated and flattened until the walls are about 1/16 inch apart inside. Then a strip of 1/16-inch sheet steel is cut the right width to fit in the flattened end of the tube, and brazed in place. The bolt holes then pass through the combined thickness of the tube and the steel strip,
- 40. giving a better bearing surface, which may be further increased by brazing on a washer. The long forks FF, which transmit the landing shocks to the springs, are naturally made of heavier material. The proper size tubing for them is 1 1/8 by 5/8 inches, this being the nearest equivalent to the 14 by 28 mm French tubing. However, this is not a stock size in this country and can only be procured by order, or it can be made by rolling out 15/16-inch round tubing. If the oval tubing can not be secured, the round can be employed instead, other parts being modified to correspond. The ends are reinforced in the same way as described for the small forks. These forks are strengthened by aluminum clamps H, Figs. 27 and 28, which keep the tubes from spreading apart. Here, of course, is another call for special castings, but a handy workman may be able to improvise a satisfactory substitute from sheet steel. On each tube there are four fittings: At the bottom, the collar M to which the fork J is attached, and above, the slide G and the clamps K and L, which limit its movement. The collar and slide should be forged, but as this may be impossible, the drawings have been proportioned for castings. The work is simple and may be done by the amateur with little experience. The projecting studs are pieces of 3/4-inch, 14- gauge steel tubing screwed in tight and pinned, though if these parts be forged, the studs should be integral. The clamps which limit the movement of the slides are to be whittled out of ash or some other hard wood. The upper clamp is held in place by four bolts, which are screwed up tight; but when the machine makes a hard landing the clamp will yield a little and slip up the tube, thus deadening the shock. After such a landing, the clamps
- 41. should be inspected and again moved down a bit, if necessary. The lower clamps, which, of course, only keep the wheels from hanging down too far, have bolts passing clear through the tubes. To the projecting lugs on the slides GG are attached the rubber tube springs, the lower ends connecting with eye bolts through the beam E. These rubber tubes, of which four will be needed, are being made by several companies in this country and are sold by supply houses. They should be about 14 inches long, unstretched, and 1 1/4 inches in diameter, with steel tips at the ends for attachment. Hub Attachments. The hubs of the two wheels are connected with the link P, with universal joints N N at each end. In case the machine lands while drifting sidewise, the wheel which touches the ground first will swing around to head in the direction in which the machine is actually moving, and the link will cause the other wheel to assume a parallel position; thus the machine can run diagonally on the ground without any tendency to upset. This link is made of the same 1- by 3/8-inch oval tubing used elsewhere in the machine. In the original Bleriot, the joints are carefully made up with steel forgings. But joints which will serve the purpose can be improvised from a 1-inch cube of hard wood and three steel straps, as shown in the sketch, Fig. 27. From each of these joints a wire runs diagonally to the bottom of the tube on the other side, with a spring which holds the wheel in its normal position. This spring should be either a rubber tube, like those described above, but smaller, or a steel coil spring. In the latter case, it should be of twenty 3/4-inch coils of No. 25 piano wire. Wheels. The wheels are regularly 28 by 2 inches, corresponding to the 700 by 50 mm French size, with 30 spokes of 12-gauge wire.
- 42. The hub should be 5 1/4 inches wide, with a 5/8-inch bolt. Of course, these sizes need not be followed exactly, but any variations will involve corresponding changes in the dimensions of the forks. The long fork goes on the hub inside of the short fork, so that the inside measurement of the end of the big fork should correspond to the width of the hub, and the inside measurement of the small fork should equal the outside measurement of the large fork. Rear Skid. Several methods are employed for supporting the rear end of the fuselage when the machine is on the ground. The first Bleriot carried a small wheel in a fork provided with rubber springs, the same as the front wheels. The later models, however, have a double U-shaped skid, as shown in Figs. 23 and 24. This skid is made of two 8-foot strips of ash or hickory 1/2 by 3/4 inches, steamed and bent to the U-shape as shown in the drawing of the complete machine. Fig. 29. Details of Framework of Bleriot Main Supporting Planes
- 43. Wings. Having completed the fuselage and running gear, the wings are next in order. These are constructed in a manner which may seem unnecessarily complicated, but which gives great strength for comparatively little weight. Each wing contains two stout ash beams which carry their share of the weight of the machine, and 12 ribs which give the proper curvature to the surfaces and at the same time reinforce the beams. These ribs in turn are tied together and reinforced by light strips running parallel to the main beams. Fig. 30. Complete Rib of Bleriot Wing and Pattern from Which Web Is Cut In the drawing of the complete wing. Fig. 29, the beams are designated by the letters B and E. A is a sheet aluminum member intended to hold the cloth covering in shape on the front edge. C, D, and F are pairs of strips (one strip on top, the other underneath) which tie the ribs together. G is a strip along the rear edge, and H is
- 44. a bent strip which gives the rounded shape to the end of the wing. The ribs are designated by the numbers 1 to 12 inclusive. Ribs. The first and most difficult operation is to make the ribs. These are built up of a spruce board 3/16 inch thick, cut to shape on a jig saw, with 3/16- by 5/8-inch spruce strip stacked and glued to the upper and lower edges. Each rib thus has an I-beam section, such as is used in structural steel work and automobile front axles. Each of the boards, or webs as they are usually called, is divided into three parts by the main beams which pass through it. Builders sometimes make the mistake of cutting out each web in three pieces, but this makes it very difficult to put the rib together accurately. Each web should be cut out of a single piece, as shown in the detail drawing. Fig. 30, and the holes for the beams should be cut in after the top and bottom strips have been glued on. The detail drawing, Fig. 30, gives the dimensions of a typical rib. This should be drawn out full size on a strip of tough paper, and then a margin of 3/16 inch should be taken off all round except at the front end where the sheet aluminum member A goes on. This allows for the thickness of the top and bottom strips. In preparing the pattern for the jig saw, the notches for strips C, D, and F should be disregarded; neither should it be expected that the jig-saw operator will cut out the oval holes along the center of the web, which are simply to lighten it. The notches for the front ends of the top and bottom strips should also be smoothed over in the pattern. When the pattern is ready, a saw or planing mill provided with a saw suitable for the work, should cut out the 40 ribs (allowing a sufficient number for defective pieces and breakage) for about $2. The builder then cuts the notches and makes the oval openings with
- 45. an auger and keyhole saw. Of course, these holes need not be absolutely accurate, but at least 3/4 inch of wood should be left all around them. Nine of the twelve ribs in each wing are exactly alike. No. 1, which forms the inner end of the wing, does not have any holes cut in the web, and instead of the slot for the main beam B, has a 1 3/4- inch round hole, as the stub end of the beam is rounded to fit the socket tube. (See Fig. 23.) Rib No. 11 is 5 feet 10 1/2 inches long, and No. 12 is 3 feet long. These can be whittled out by hand, and the shape for them will be obvious as soon as the main part of the wing is put together. The next step is to glue on the top and bottom strips. The front ends should be put on first and held, during the drying, in a screw clamp, the ends setting close up into the notches provided for them. Thin 1/2-inch brads should be driven in along the top and bottom at 1- to 2-inch intervals. The rear ends of the strips should be cut off to the proper length and whittled off a little on the inside, so that there will be room between them for the strip G, 1/4 inch thick. Finally, cut the slots for the main beams, using a bit and brace and the keyhole saw, and the ribs will be ready to assemble. Beams and Strips. The main beams are of ash, the front beam in each wing being 3 1/4 by 3/4 inches and the rear beam 2 1/2 by 5/8 inches. They are not exactly rectangular but must be planed down slightly on the top and bottom edges, so that they will fit into the irregularly-shaped slots left for them in the ribs. The front beams, as mentioned above, have round stubs which fit into the socket tube on the fuselage. These stubs may be made by bolting
- 46. short pieces of ash board on each side of the end of the beam and rounding down the whole. To give the wings their slight inclination, or dihedral angle, which will be apparent in the front view of the machine, the stubs must lie at an angle of 2 1/2 degrees with the beam itself. This angle should be laid out very carefully, as a slight inaccuracy at this point will result in a much larger error at the tips. The rear beams project about 2 inches from the inner ribs. The ends should be reinforced with bands of sheet steel to prevent splitting, and each drilled with a 3/8-inch hole for the bolt which attaches to the fuselage strut. A strip of heavy sheet steel should be bent to make an angle washer to fill up the triangular space between the beam and the strut; the bolt hole should be drilled perpendicularly to the beam, and not to the strut. The outer ends of the beams, beyond rib No. 10, taper down to 1 inch deep at the ends. The aluminum member A, Fig. 29, which holds the front edge of the wing in shape, is made of a 4-inch strip of fairly heavy sheet aluminum, rolled into shape round a piece of half-round wood, 2 1/4 inches in diameter. As sheet aluminum usually comes in 6-foot lengths, each of these members will have to be made in two sections, joined either by soldering (if the builder has mastered this difficult process) or by a number of small copper rivets. No especial difficulties are presented by the strips, C, D, and F, which are of spruce 3/16 by 5/8 inch, or by the rear edge strip G, of spruce 1/4 by 1 1/2 inches. Each piece H should be 1 by 1/2 inch half-round spruce, bent into shape, fitted into the aluminum piece at the front, and at the rear flattened down to 1/4 inch and reinforced by a small strip glued to the back, finally running into the strip G.
- 47. The exact curve of this piece does not matter, provided it is the same on both wings. Assembling the Wings. Assembling the wings is an operation which demands considerable care. The main beams should first be laid across two horses, set level so that there will be no strain on the framework as it is put together. Then the 12 ribs should be slipped over the beams and evenly spaced 13 inches apart to centers, care being taken to see that each rib stands square with the beams, Fig. 31. The ribs are not glued to the beams, as this would make repairs difficult, but are fastened with small nails. Strips C, D, and F, Fig, 29, are next put in place, simply being strung through the rows of holes provided for them in the ribs, and fastened with brads. Then spacers of 3/16-inch spruce, 2 or 3 inches long, are placed between each pair of strips halfway between each rib, and fastened with glue and brads. This can be seen in the broken-off view of the wing in the front view drawing, Fig. 23. The rear edge strip fits between the ends of the top and bottom strips of the ribs, as mentioned above, fastened with brads or with strips of sheet-aluminum tacked on.
- 48. Fig. 31. Assembling the Main Planes of a Bleriot Monoplane Each wing is trussed by eight wires, half above and half below; half attached to the front and half to the rear beam. In the genuine Bleriot steel tape is used for the lower trussing of the main beams, similar to the tape employed in the running gear, but American builders prefer to use 1/8-inch cable. The lower rear trussing should be 3/32- or 7/64-inch cable, and the upper trussing 3/32-inch. The beams are provided with sheet-steel fixtures for the attachment of the cables, as shown in the broken-off wing view, Fig. 23. These are cut from fairly-heavy metal, and go in pairs, one on each side of the end beam, fasten with three 3/16-inch bolts. They have lugs top and bottom. They are placed between the fifth and sixth and ninth and tenth ribs on each side. To resist the backward pressure of the air, the wings are trussed with struts of 1-inch spruce and 1/16-inch cable, as shown in Fig.
- 49. 23. The struts are placed between the cable attachments, being provided with ferrules of flattened steel tubing arranged to allow the rear beam freedom to swing up and down. The diagonal cables are provided with turnbuckles and run through the open spaces in the ribs. Control System. The steering gear and tail construction of the Bleriot are as distinctive as the swiveling wheels and the U-bolts, and the word cloche applied to the bell-like attachment for the control wires, has been adopted into the international vocabulary of aeroplaning. The driver has between his knees a small steering wheel mounted on a short vertical post. This wheel does not turn, but instead the post has a universal joint at the bottom which allows it to be swung backward and forward or to either side. The post is really a lever, and the wheel a handle. Encircling the lower part of the post is a hemispherical bell—the cloche—with its bottom edge on the same level as the universal joint. Four wires are attached to the edge of the cloche. Those at the front and back are connected with the elevator, and those at the sides with the wing-warping lever. The connections are so arranged that pulling the wheel back starts the machine upward, while pushing it forward causes it to descend, and pulling to either side lowers that side and raises the other. The machine can be kept on a level keel by the use of the wheel and cloche alone; the aviator uses them just as if they were rigidly attached to the machine, and by them he could move the machine bodily into the desired position. In practice, however, it has been found that lateral stability can be maintained more easily by the use of the vertical rudder than by warping. This is because the machine naturally tips inward on a turn,
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