Three‐color immunofluorescence analysis of leu antigens on human peripheral blood using two lasers on a fluorescence‐activated cell sorter

A fluorescence-activated cell sorter was modified to quantify simultaneously three immunofluorescence stains on a population of cells. A dye laser containing rhodamine 6G was used to obtain 600 nm light to excite Texas Red coupled avidin. An argon ion laser operating at 488 nm excited both fluorescein and phycoerythrin directly conjugated antibodies. The emission from these fluorophores could be independently quantified as demonstrated by the histograms generated by samples labeled separately with each of the three stains. This three-color detection system was used to analyze human peripheral blood mononuclear cells for the expression of three antigens: Leu 2, Leu 7, and Leu 11. Upon reanalysis of the list mode data, several discrete subpopulations of lymphocytes could be identified based on the quantitative expression of these three antigens. Of particular interest in the normal sample studied was a population of dimly labeled Leu 2+ cells which were predominantly Leu 11+, a phenotype which is seen infrequently in normal individuals. This technique expands the combinations of antigens that can be studied at any one time and will facilitate the detection and functional analysis of cells in heterogeneous populations.