FLOW cytometry is new to me. Could someone please describe what the attached result image is showing?
Thanks!
BY

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Attached file: flowpicture.png (71 downloads, 7KB)

BY said:

FLOW cytometry is new to me. Could someone please describe what the attached result image is showing? Thanks! BY

Flow Cytometry or FACS(Fluorescent Activated Cell Sorter) provides the most powerful and effective means of fractionating cell populations. Cells suspensions are treated with an antigen or antibody labelled with a fluorochrome reagent . A liquid stream containing the dilute suspension of cells passes through a small orifice which is vibrated to break up the lquid into regularly spaced droplets of uniform size ( usually no more than one cell per droplet). Cells are irradiated with a high intensity beam of light from a laser. The degree of light scatter and intensity of light emitted by the laser-activated fluorochrome is measured by a pair of photodetectors and the information processed by a computer.
Results could be plotted in form of histograms, overlaying positively charged cells ( shifted to the right) with the negative cells or controls( histogram at the left side of the picture).

The histogram plot that you have is a composite anaysis of a few hundred to a few thousand individual "events" or cells that have singly passed in front of the laser, with physical and fluorescence properties being measured by a series of photo-multiplier tubes (PMTs). In most cytometers, data is measured across a series of "channels" (usually 1024) across multiple parameters. These include physical properties such as forward scatter (indicative of cell size), side scatter (indicative of internal/organelle complexity within the cell), and multiple fluorescence channels (usually Fl1, 2, etc) depending upon emission wavelengths of the fluorophore(s) used. In a "single-parameter" histogram plot, one usually sees the fluorescence intensity (increasing along x-axis) v/s the total number of cells at any given intensity ("counts").

Forgot to mntion - the two plots in your histogram overlay possibly represent a control flourescence sample (lower fluorecence) (can be cells alone, secondary antibody alone, or a non-specific antibody/rgt) and an actual experimental sample. This gives direct visual inference that your experiment sample has an enhanced *specific* fluorescence over the control. These plots can be "gated" or "subtracted" to obtain statistical analyses.