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FACS

The FACS Technique

Resting lymphocytes comprise many functional subpopulations, which are identified and distinguished from each other on the basis of heir differential expression of cell surface proteins, which can be detected using specific antibodies.

Flow Cytometry

A powerful tool for defining and quantifying lymphocytes is the flow cytometer, which detects and counts individual cells passing in a stream through a laser beam. A flow cytometer equipped to separate the identified cells is called a fluorescence-activated cell sorter (FACS). These instruments are used to study the properties of cell subsets identified using monoclonal antibodies to cell-surface proteins. Individual cells within a mixed population are first tagged by treatment with specific monoclonal antibodies labeled with fluorescent dyes, or by specific antibodies followed by labeled anti-immunoglobulin antibodies. The mixture of labeled cells is then forced with a much arger volume of saline through a nozzle, creating a fine stream of liquid containing cells spaced singly at intervals . As each cell passes through a laser beam it scatters the laser light, and any dye molecules bound to the cell will be excited and will fluoresce.

Sensitive photomultiplier tubes detect both the scattered light, which gives information on the size and granularity of the cell, and the florescence emissions, which give information on the binding of the labeled monoclonal antibodies and therefore on the expression of cell-surface proteins by each cell. In the cell sorter, the signals passed back to the computer are used to generate an electric charge, which is passed from the nozzle through the liquid stream at the precise time that the stream breaks up into droplets, each containing no more than a single cell; droplets containing a charge can then be deflected from the main stream of droplets as they pass between plates of opposite charge, so that positively charged droplets are attracted to a negatively charged plate, and vice versa. In this way, specific subpopulations of cells, distinguished by the binding of the labeled antibody, can be purified from a mixed population of cells. Alternatively, to deplete a population of cells, the same fluorochrome can be used to label different antibodies directed at marker proteins expressed by the various undesired cell types.

facs

Cell Sorting

The cell sorter can be used to direct labeled cells to a waste channel, retaining only the unlabeled cells. When cells are labeled with a single fluorescent antibody, the data from flow cytometer are usually displayed in the form of a histogram of fluorescence intensity vs. cell numbers. If two or more antibodies are used, each coupled to different fluorescent dye, then the data are more usually displayed in he form of a two-dimensional scatter diagram or as a contour diagram, where the fluorescence of one dye-labeled antibody is plotted against that of a second, with the result that a population of cells labeling with one antibody can be further subdivided by its labeling with the second antibody. By examining large numbers of cells, flow cytometry can give quantitative data on the percentage of cells bearing different molecules. As the power of FACS technology has grown, progressively more antibodies labeled with distinct fluorescent dyes can be used at the same time.

cell sorting

Three, four and even five color analyses can now be handled by very powerful machines

FACS Protocols

 

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