Fluorescence-activated cell sorting of transfected cells

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This chapter discusses the fluorescence-activated cell sorting of transfected cells. Fluorescence-activated cell sorting (FACS) is a technique that can measure the fluorescence intensity of individual cells within a population. The cells within a population are labeled with a fluorescent label in such a way that the intensity of fluorescence of an individual cell provides a quantitative measure of the particular parameter of interest. By staining cells with fluorochromes of different emission wavelengths, multiple parameters can be measured simultaneously. FACS has a number of applications within cell cycle research. For example, cellular DNA content is commonly measured with a fluorescent DNA intercalating agent such as propidium iodide (PI). The study of putative cell cycle regulatory proteins often requires their activities within the cell to be either enhanced or inhibited, and the consequences observed in terms of, for example, changes in cell cycle distribution.

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      A proper FACS analysis requires measurements of nontransfected cells and determination of cell death to ensure accurate measurements of relative surface and total cell receptor levels. A more complete description of FACS analysis of transfected cells can be found elsewhere (Adams, Lopez, Sellers, & Kaelin, 1997). Split a 10-cm tissue culture dish of confluent HEK293 cells 1:10 in DMEM with 10% FBS.

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      However, it usually requires a population of cells more than 100,000 in order to achieve a high yield, which limits its applications in small-scale cell sorting such as isolation of rare cells. For example, transient gene transfection of cells in a 96-well dish usually yields less than 100,000 cells with a 5–30% efficiency [4]. The resulting cells need to be purified before further studies of gene function and expression can be conducted.

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