Flow Cytometry

Cells can be sorted into different classes using a fluorescence activated cell sorter (FACS). For this, cells are selectively labeled with a fluorescent dye and sorted into classes based on the extent of labeling. As described above, fluorescence labeling can be done using an antibody-conjugated fluorescent dye or a fluorescent dye such as DAPI that binds a specific cellular component. For example, an antibody specific to a particular cell surface protein is conjugated to a fluorescent dye. This will bind to the surface of cells that express the surface protein and these cells will be fluorescent. Cells lacking this surface protein will not be fluorescent. The extent of the fluorescence can also be quantified and the cells sorted based on the amount of fluorescence.

In the FACS, cells treated with the fluorescent dye label flow past a laser beam and will or will not fluoresce based on the level of bound dye. The cells are then dispersed into droplets containing no more than one cell. Droplets containing a fluorescent cell are made negatively charged and the charge is used to separate the droplets into categories. The number of cells in a category can be quantified and those cells falling into a specific category can be separated from the remainder of the cells and used for further analysis by other methods. DNA staining with a fluorescent dye is used to distinguish cells in G1 of the cell cycle containing the haploid amount of DNA (1 C amount of DNA) from cells in G2 (2C) from cells in the S phase (1-2C). Figure 2.7 shows such an analysis. Cells containing the temperature-sensitive mutation mcm2-l arrest at the nonpermissive temperature with 2C DNA content while wild-type cells continue to proceed through the cell cycle (evidenced by a distribution of cells with 1 C or 2 C DNA content or amounts in between the two). Also, Figure 2.7 shows that normal cell cycling is restored in the mcm2-l dbf4-6 double mutant strain. This is an example of suppression (see Chapter 8).

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