Fig. A.21 Flow cytometers allow individual cells to be identified by their cell-surface antigens and to be sorted. Cells to be analyzed by flow cytometry are first labeled with fluorescent dyes (top panel). Direct labeling uses dye-coupled antibodies specific for cell-surface antigens (as shown here), while indirect labeling uses a dye-coupled immunoglobulin to detect unlabeled cell-bound antibody. The cells are forced through a nozzle in a single-cell stream that passes through a laser beam (second panel). Photomultiplier tubes (PMTs) detect the scattering of light, which is a sign of cell size and granularity, as well as emissions from the different fluorescent dyes. This information is analyzed by computer (CPU). By examining a large number of cells, the proportion of cells with a specific set of characteristics can be determined and levels of expression of various molecules on these cells can be measured. The lower part of the figure shows how these data can be represented, the example in this case being the expression of two surface immunoglobulins, IgM and IgD, on a sample of B cells from a mouse spleen. The two immunoglobulins have been labeled with different-colored dyes. When the expression of just one type of molecule is to be analyzed (IgM or IgD), the data are usually displayed as a histogram, as in the left-hand panels. Histograms display the distribution of cells expressing a single measured parameter (for example, size, granularity, fluorescence intensity). When two or more parameters are measured for each cell (IgM and IgD), various types of two-dimensional plots can be used to display the data, as shown in the right-hand panel. All four plots represent the same data, and in each case, the horizontal axis represents intensity of IgM fluorescence, and the vertical axis the intensity of IgD fluorescence. Two-color plots provide more information than histograms; they allow recognition, for example, of cells that are ‘bright’ for both colors, ‘dull’ for one and bright for the other, dull for both, negative for both, and so on. For example, the cluster of dots in the extreme lower left portions of the plots represents cells that do not express either immunoglobulin, and are mostly T cells. The standard dot plot (upper left) places a single dot for each cell whose fluorescence is measured. This format works well for identifying cells that lie outside the main groups, but tends to saturate in areas containing a large number of cells of the same type. A second means of presenting these data is the color dot plot (lower left), which uses color density to indicate high-density areas. A contour plot (upper right) draws 5% ‘probability’ contours, with contour lines drawn to indicate each successive 5% of the population; this format provides the best monochrome visualization of regions of high and low density. The lower right plot is a 5% probability contour map, which also shows outlying cells as dots.