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FLOW CYTOMETRY

What the assay tells you

Flow cytometry can be used to count the number of cells in a suspension having specific molecules on their membrane or (with fixation and permeabilization) in their cytosol. It will also quantify the relative number of molecules on each cell. Fluorochrome-labeled antibody is used to bind specific molecules. When illuminated by a laser emitting UV light, the fluorochrome emits visable light in a specific wavelength that can be detected by a photomultiplier tube.

What you need to do the assay

• Cell suspension in dust-free buffer.
• Fluorochrome-labeled antibody to membrane marker or cytokine.
• Standard solutions (positive and negative controls).
• Flow cytometer.

How the assay is done

• If the assay is being done to quantify cytokine production, incubate the cytokine-producing cells in a drug to block secretion. Then fix the cells (formalin) to cross link the cytokine and keep it inside the cell, followed by treatment with detergent to permeablize the cell (open the cell membrane) so that antibodies can enter and bind the cytokine. This step is not necessary if the assay is to quantify external membrane molecules.
• Incubate the cell suspension with desired antibodies; wash. Several antibodies labeled with different fluorochromes may be used simultaneously as long as they have distinct specificities. Common fluorochromes are FITC (fluorescein isothiocyanate, green) and PE (phycoerythrin, red.)
• Run cells through flow cytometer (above). Vibrating nozzle breaks narrow stream of cells into droplets, each containing one cell. As cells pass by laser beam and detector, the amount of fluorescence on each cell is quantified and converted to electronic signals that can be recorded and plotted using a computer program. Forward and side scatter of light can also be analyzed to measure cell size and granularity.

How to interpret the results

For one color fluorescence, fluorescence intensity (amount of fluorescence) is plotted on a log scale on the horizontal axis (see Histograms above). Numbers of cells at each fluorescence intensity are plotted on the vertical axis. The area under the curve represents the total number of cells analyzed. The further to the right the mean fluorescence intensity, the more marker or cytochrome is present on the cell population.

For two color fluorescence, fluorescence intensity for one fluorochrome-tagged antibody is plotted on a log scale on the vertical axis and fluorescence intensity for the other fluorochrome-tagged antibody is plotted on a log scale on the horizontal axis (see Dot Plot above) . Fluorescence intensity for each cell is shown by a dot whose vertical and horizontal position indicates fluorescence intensity for each fluorochrome-tagged antibody. Plots are usually divided into four areas. The bottom left area shows cells which bind neither antibody (double negative). The top right area shows cells which bind both antibodies (double positive). The top left area shows cells which bind only the antibody shown on the vertical axis (single positive). The bottom right area shows cells which bind only the antibody shown on the horizontal axis (single positive).

Additional comments

A fluorescence-activated cell sorter (FACS) can use the fluorescence signals to polarize the drops and sort the cells into as many as three fractions: for example, cells binding FITC-antibody, cells binding PE-antibody, and cells binding neither antibody.

Useful web sites

Flow cytometry: http://biology.berkeley.edu/crl/flow_cytometry_basic.html

http://www.wehi.edu.au/cytometry/flowintrol.html

CD antigens: http://www.beckmancoulter.com/products/instrument/flowcytometry/ecatalog/cdchart.asp

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