Basics of Flow cytometer and its potential applications.doc
- 1. Basics of Flow cytometry and its potential applications Ajay Kumar, Division of Biochemistry, IVRI, Izatnagar, Bareilly, India Flow cytometry is a widely used method for analyzing the expression of cell surface proteins and intracellular molecules and organelles function like mitochondrial membrane potential and identifying different cell types in a heterogeneous cell population. It allows simultaneous multi-parameter analysis of single cells. It is predominantly used to measure fluorescence intensity produced by fluorescent-labeled antibodies for detecting proteins, or ligands that bind to specific cell-associated molecules such as propidium iodide binding to DNA. The staining procedure involves making a single-cell suspension from cell culture or tissue samples. The cells are then incubated with fluorochrome-labeled antibodies and analyzed on the flow cytometer (FCM). During acquisition, cells are focused into a one-cell- wide beam by a fluidics setup. The beam of cells is analyzed through light scattering techniques i.e. forward scatter (FSC), proportional to the size of each cell, and side scatter (SSC), proportional to cell complexity or number of internal compartments which give information on the size and composition of each cell. Leukocyte sub-populations can be determined using scattering techniques (especially for determining between lymphocytes, monocytes and neutrophils) as shown below. The other detection techniques rely on fluorescence. Excitation sources within the cytometer (usually lasers) excite fluorophores in probes attached to the cells, and several detectors measure the resulting emitted photons. A flow cytometer consists of four basic components: fluidic system, laser(s), optics, and electronics. Additionally, a sorting device can be added to collect cells after they have run past the laser. Fluidic system: The flow chamber, as the core of the flow cytometer, transports particles (cells or microbes) to the laser. The interrogation point where the laser intersects with the particles, is also the place the optics system detects light scatter and fluorescence. The fluidic system of a flow cytometer is designed to deliver cells in an orderly single file stream to the interrogation point so that only one cell passes through at a time, through a process called hydrodynamic focusing. Laser: FCM is generally equipped with minimum of one laser i.e. Argon laser which emits a 488 nm light, can excite multiple fluorochromes another red laser (emitting a 635nm light) can be added to increase the number of parameters to be studied. Various fluorochromes can
- 2. be used in combination if each is excited by 488 nm and their peak emission wavelengths are not too close to each other. Two of the most widely used fluorochromes that meet these criteria are fluorescein isothiocyanate (FITC) and phycoerythrin (PE). The absorption spectra of FITC and PE peak at approximately 495 nm and are easily excited by the argon laser's 488 nm wavelength Optics: The optical system of a flow cytometry consists of two parts: excitation optics and collection optics. The excitation optics is through lasers that consistently intersect with particles. Light emitted from the particle is collected with a lens (collection lens) and passed through a system of optical mirrors (dichroic) and filters (bandpass, shortpass, or longpass) which reroute specified wavelengths of light to designated optical detectors. This process is achieved by the collection optics. The detectors convert the light signals into currents, which are then sent to the electronics system. Electronics: The function of the electronics system of the flow cytometer is twofold; convert light signals into electronic signals (voltages) and perform data analysis. The former is achieved by one of the two types of photodetectors mentioned above (photodiodes and photomultiplier tubes). Once the data is resulted, next is to analyze the data output through 1) Histogram and 2) Dot plots. A histogram is a single parameter plot where the y-axis shows the number of events (cell count) and the x-axis (log or linear) depicts the parameters digital signal value while in dot plots, two-parameter graphical representation of particle's properties can be achieved where each point plotted corresponds to an individual cell. Histogram image of flow cytometer Dot plot image of flow cytometer In this picture of dot plot, upper left quadrant shows 23.0% and 32.5% in control and vaccinated group resp. means CD4 positive cells while lower right quadrant shows 17.8% and 17.0% in control and vaccinated group resp. means CD8 positive cells and their ratio
- 3. (CD4:CD8 positive cells) are 1.29 (23.0/17.8) in control while in vaccinated group, it is 1.91 (32.5/17.0). One of the important applications of flow cytometer in pharmacological research is to see the effect of toxins or drugs on the health of mitochondria through mitochondrial membrane potential (MMP). JC-1 dye is being used to measure the MMP. The membrane-permeant JC- 1 dye is widely used in apoptosis studies to monitor mitochondrial health. JC-1 dye exhibits potential-dependent accumulation in mitochondria, indicated by a green fluorescence emission at (~529 nm) for the monomeric form of the dye, which shifts to red (~590 nm) with a concentration-dependent formation of red fluorescent J-aggregates (see Figure 1). Consequently, mitochondrial depolarization is indicated by a decrease in the red/green fluorescence intensity ratio. Protocol for JC-1 dye for MMP by flow cytometer: 1. Take around 106 single cell suspension either from tissue origin or cell line in a microfuge tube and add 750ul PBS. 2. Divide the cells in three tubes of 250ul each. 3. One tube will be kept as unstained control. 4. To second tube, add 50μM final concentration of Carbonyl cyanide 3- chlorophenylhydrazone (CCCP) and incubate the cells at 37°C for 5 minutes (as a positive control). CCCP, a chemical inhibitor of oxidative phosphorylation, affecting the protein synthesis reactions in mitochondria and causing the gradual destruction of living cells and to it add 10 μl of 200 μM JC-1 dye (2 μM final concentration) and incubate the cells at 37°C, 5% CO2 for 15-30 min. 5. To third tube, add 10 μl of 200 μM JC-1 dye (2 μM in final concentration) and incubate the cells at 37 °C, 5% CO2 for 15-30 min. 6. Wash all samples by adding 1 ml of warm PBS at 37°C and centrifuge for 5 min at 25°C at 400 × g.. 7. Remove the supernatant and resuspend the cell pellet again in 300 μl fresh cell culture medium or PBS. 8. Acquire all three samples in flow cytometer and analyze on a flow cytometer with 488 nm excitation using emission filters appropriate for green and red fluorescein (FL-1 and FL-2 filter respectively). Mitochondrial membrane potential study by Flow cytometer
- 4. (Tim Bushnell, 2018) Various other applications of Flow Cytometry: 1. Cell cycle analysis 2. Cell viability and apoptosis study 3. Morphological complexity of cells 4. Cellular uptake of synthetic peptides 5. Membrane fluidity and potential 6. Oxidative stress study 7. Protein expression and localization 8. Chromosome analysis 9. Sex preselection through sperm sorting 10. Immunophenotyping of cells 11. Biomarkers based tumor characterization Recommended readings: Flow Cytometry: A Practical Approach, 3rd Edition. M.G. Ormerod, Oxford University Press (2000) Flow Cytometry: Clinical Applications. Marion G. Macey. Blackwell Scientific Publications, Oxford (1994)