Analysis of single and double-stained alveolar macrophages by flow cytometry

The quantification of cell surface antigens on human alveolar macrophages using flow cytometry is complicated by strong autofluorescence which varies with cell size and granularity. We report here a new method for overcoming the analytical problems caused by autofluorescence. After positioning the unstained cells along the 0, 0; 10(4), 10(4) diagonal on all three fluorescence dot-plots (FL1 vs. FL2; FL2 vs. FL3; FL1 vs. FL3) of a single-laser flow cytometer (excitation wavelength at 488 nm) and adjusting compensation so that the reference FL3 channel profile is not changed by PE-staining, the cell population on the FL3 histogram is arbitrarily classified into subpopulations having similar autofluorescence intensity. These are subsequently back-gated onto the FL1 vs. FL2 dot-plot and separately analyzed. The percentage of stained cells in the whole population is then calculated on the basis of absolute numbers or as the weighted mean of all the subpopulations. This approach permits the analysis not only of single-stained but also double-stained human alveolar macrophages.