How Works the Technique of Flow Cytometry - Research Paper Example

? Flow Cytometry By College Flow Cytometry Principle: The technique of flow cytometry is based on the principles of light scattering, excitation and emission by fluorochrome tagged cells, when they are hydro-dynamically focused through a sheath and are intercepted by an a light source. The scattered light, gives information on the size and granularity of the cell, and the fluorescence emission gives information on the binding of the labeled monoclonal antibodies and hence on the expression of cell surface proteins by each cell. The fluorochrome is bound to an antibody and this antibody bids to the cells. The light source usually used is a laser light. The cells are hydro-dynamically maneuvered such that only one cell intercepts the laser beam at a given time. When these cells intercept the laser beam, light is scattered by them and absorbed by the fluorochrome. The fluorochrome gets excited and releases a photon of light with specific spectra unique to that fluorochrome. This scattered and emitted light is converted to electrical pulses by the optical detectors. Parallel light waves are picked up by the confocal lenses focused at the point where the cells intercept the laser beam. Optical filters are used to send light to different detectors, where it is processed by a series of linear and log amplifiers and finally the analogs are digitalized to plots and graphs (Berkeley). Flow cytometry is done when the experimenter is not interested in keeping the cells but simply in analyzing the distribution of cell size and/or surface molecules on the cells in the suspension. But if he/she is interested in identifying and separating the cells, then a flow cytometer equipped with fluorescence activated cell sorter (FACS) is required. In FACS, the scattered and emitted signals passed onto the computer are used to generate electrical charge, which is then passed onto the cells-stream just before forming droplets. Having attained a charge, these cells deflect from the main stream as they pass between plates having opposite charge. Positive drops go to negatively charged plate and vice versa. In this way subpopulation of cells can be purified from a collection of cells, distinguished by the labeled antibodies. Data Analysis: An important principle of flow cytometry data analysis is to selectively visualize the cells of interest while eliminating results from unwanted particles e.g. dead cells and debris. This procedure is called gating. This is based on the fact that dead cells have lower forward scatter and higher side scatter than living cells. The data collected by the computer is represented in several ways like density plots, contour diagrams, and histograms. On density plots each dot represents an individual cell that has intercepted the laser beam. Contour diagrams are an alternative way to demonstrate the same data. Joined lines represent similar numbers of cells. Histograms can be a single parameter or two parameter histograms based on the parameters displayed on the two axes. Single parameter histograms are graphs that display a single measurement parameter (relative fluorescence or light scatter intensity) on the x-axis and the number of events (cell count) on the y-axis. Two parameter histograms are graphs that display two measurement parameters, one on the x-axis and one on the y-axis, and the cell count as a density (dot) plot or contour map (Kenneth 2012). Technical Hints: Single cells must be suspended at a density of 105–107 cells/ml to prevent tubing from clogging up. The rate of flow sorting should be adjusted to 2000–20,000 cells/second. If cells have intracellular antigens, these cells have to be fixed and permeabilized prior to adding the fluorochrome. This allows probes to access intracellular structures while leaving the morphological scatter characteristics of the cells intact. Splenocytes: Mouse spleen can have several cell types at any given time. These cells could be B cells and its subtypes, T cells and its subtypes, dendritic cells, monocytes, macrophages, myeloid cells, fibroblasts, bone marrow derived dendritic cells etc. These cells can be in different stages of differentiation and hence express different cell surface markers. Monoclonal antibodies (mAb) against these markers can be used to separate different cell types from splenocytes using multi-parameter FACS analysis (Hsueh, Roach, Lin and O'Connell, 2002). The B cells having CD19 and T cells having CD3 (Kindt, Osborne and Goldsby, 2006), can be separated using the respective anti-CD19 and anti-CD3 monoclonal antibodies. Since follicular dendritic cells (also present amongst splenocytes) also express CD19, they can be separated from mature B cell by using anti-body specific for them, which is FDC-M2 (Taylor et al., 2002). Also, since B220 is present in subsets of NK cells, activated T cells, and a subpopulation of dendritic cells, CD19 is preferred to separate B cells from T cells instead of B220 (Rolink et al., 1996). There are two types of mature B cell types in the mouse spleen; follicular (FO) B cells and the marginal zone (MZ) B cells. CD36, CD68, and CD49e are expressed on MZ B cells but not on FO B cells. The authors have concluded that CD36 can be used to distinguish these two cell types (Zhang et al., 2007). CD9 (Won and Kearney 2002) can also be used to distinguish between MZ B cells from FO B cells. Hence anti-CD36 or anti-CD9, which is expressed on MZB cells, can be used to separate them from FO B cells. The other types of B cells found in the mouse spleen are B1 and B2 type B cells. B2 cells express B220 and high levels of CD23.  B1 cells express B220 but have little or no CD23. Hence levels of anti-B220 and anti-CD23 can be used to separate type B2 from type B1 and transitional immature B cells. There are two types of T cells in the spleen; CD4+ (T helper, Th) and CD8+ (cytotoxic T cells) (Playfair and Chain, 2001). These two can be separated using respective anti-CD4 and anti-CD8 mAbs. Using both the antibodies together in FACS, double negative, CD4--/CD8--, (DN) T cells can be separated from the two. Subsets of T helper cells can also be separated using specific antibodies. There are three types of T helper (CD4+) cells. Regulatory T cells have specific marker called Neuropilin-1 (Bruder et al, 2004), Follicular helper T cells have CXCR5 (Poholek et al 2010), and Th17 have IL-17A (Uyttenhove and van Snick 2006). These three subsets of CD4+ can be separated using anti-neuropili-1, anti-CXCR5, and anti-IL-17A monoclonal antibodies. Table 1: Cell type and corresponding mAb that can be used to separate them. Cell Type mAb against these antigens B cells CD19 Marginal zone B cells CD36, CD9 B2 B cells CD23 T cells CD3 Helper T (Th) cells CD4 Regulatory Th cells Neuropilin-1 Follicular Th cells CXCR5 Th17 Th cells IL-17A Cytotoxic T cells CD8 Reference List Berkeley. Basic Infromation About Flow Cytometry and Compensation. [Online]. Flow Cytometry Facility. Available at: [Accessed on 22 June 2012]. Bruder, D., Probst-Kepper, M., Westendorf, A.M., Geffers, R., Beissert, S., Loser, K., von Boehmer, H., Buer, J., and Hansen, W., 2004. Neuropilin-1: a surface marker of regulatory T cells. Eur J Immunol, 34(3), pp. 623-30. Hsueh, R.C., Roach, T. I. A., Lin K., O'Connell, T,D., Heping, H., Zhen, Y., 2002. Purification and Characterization of Mouse Splenic B Lymphocytes. [Online]. AFCS Research Reports, 1(1). Available at: < http://afcs.lbl.gov/reports/v1/BC00[01/BC0001.htm>. [Accessed on 22 June 2012]. Kenneth, M., 2012. Janeway’s Immuno Biology. 8th ed. New York: Garland Science, Taylor & Fransis Group, LLC. Kindt, T.J., Osborne, B.A,and Goldsby, R.A., 2006. Kuby Immunology. 6th ed. W.H. New York: Freeman & Company. Playfair, J.H.L. and Chain, B.M., 2001. Immunology at a Glance. 7th ed. Malden: Blackwell Publishing. Poholek, A.C., Hansen, K., Hernandez, S.G,, Eto, D., Chandele, A., Weinstein, J.S., Dong, X., Odegard, J.M., Kaech, S.M., Dent, A.L., Crotty, S., and Craft, J., 2010. In vivo regulation of Bcl6 and T follicular helper cell development. J Immunol, 185(1), pp. 313-26. Rolink, A., ten Boekel, E., Melchers, F., Fearon, D.T., Krop, I., and Andersson, J., 1996. A subpopulation of B220+ cells in murine bone marrow does not express CD19 and contains natural killer cell progenitors. J Exp Med, 83(1), pp. 187-94. Taylor, P.R., Pickering, M.C., Kosco-Vilbois, M.H., Walport, M.J., Botto, M., Gordon, S., and Martinez-Pomares, L., 2002.The follicular dendritic cell restricted epitope, FDC-M2, is complement C4; localization of immune complexes in mouse tissues. Eur J Immunol, 32(7), pp. 1888-96. Uyttenhove, C. and van Snick, J., 2006. Development of an anti-IL-17A auto-vaccine that prevents experimental auto-immune encephalomyelitis. Eur J Immunol, 36(11), pp. 2868-74. Won, W.J., and Kearney, J.F., 2002. CD9 is a unique marker for marginal zone B cells, B1 cells, and plasma cells in mice. J Immunol, 168(11), pp.5605-11. Zhang, P., Li, W., Wang, Y., Hou, L., Xing, Y., Qin, H., Wang, J., Liang, Y., and Han, H., 2007. Identification of CD36 as a new surface marker of marginal zone B cells by transcriptomic analysis. Mol Immunol, 44(4), pp. 332-7. Read More