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On Flow Cytometry - Essay Example

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Flow Cytometry By Your Name Tutor name College name 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…
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Essay on Flow Cytometry
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"On Flow Cytometry"

Download file to see previous pages 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 sub ...Download file to see next pagesRead More
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