Labeling Cells for use in Multicolor Color Flow Cytometry
Cells can be simultaneously examined for expression of up to 4 different molecules, as long as the mAbs used are of different isotype. Isotype-specific 2nd step antibodies conjugated to 4 different fluorochromes can be used to visualize the pattern of expression of two or more molecules expressed on the same or different cell subsets. Multicolor labeling provides a way to analyze the composition of complex populations of leukocytes. It also provides a way to minimize the number of samples needed for analysis. The protocol for labeling is the same as for single flow cytometry. At present, there are few directly conjugated 2nd step reagents for use in species other than humans and laboratory animal species so indirect labeling is necessary. Each mAb used in indirect multicolor flow cytometry must be of a different isotype. For multicolor analysis, it is convenient to prepare cocktails of mAbs so that all antibodies are present at 15 mg/ml in a single solution. This speeds up the process of making up labeling plates, and also keeps the volume in each well from getting too large. Cocktails of 2nd step reagents can be prepared also at the time of use.
Equipment, supplies and solutions are the same as indicated for single fluorescence labeling. The additional reagents needed are isotype-specific anti-mouse immunoglobulin antibodies conjugated to different fluorochromes, typically FITC and PE. Isotype-specific reagents are available from Southern Biotechnology, as well as other commercial sources.