Measurement of lymphocyte subset proliferation by three-color immunofluorescence and DNA flow cytometry

We developed a method for simultaneous flow cytometric analysis of three-color immunofluorescence and DNA content. We show here that staining with 7-amino-actinomycin D (7-AAD) at 10 microg/ml using a phosphate-citrate buffer at low pH containing saponin for cell membrane permeabilization yields good resolution DNA histograms with low coefficients of variation. Furthermore, light scatter properties of cells are preserved after permeabilization; this permits gating on cell populations that differ in scatter signals on the flow cytometer. Because of the low pH of the phosphate-citrate staining buffer, Alexa488, a pH-independent green-fluorescent fluorochrome is used instead of fluorescein-isothiocyanate (FITC) for cell surface staining in combination with phycoerythrin (PE) and with allophycocyanin (APC) which are both pH insensitive. Removal of 7-AAD after staining and replacing it with non-fluorescent actinomycin D (AD) retains DNA staining and allows detection of Alexa488, PE and APC cell surface immunofluorescence without interference from fluorescent 7-AAD in solution for clear identification of cell subpopulations even after prolonged stimulation in culture. Thus, using a four-color benchtop flow cytometer, measurement of Alexa488, PE and APC three-color immunofluorescence can be combined with 7-AAD DNA content analysis. Furthermore, we demonstrate that sample storage overnight without fixation for later analysis on the flow cytometer is possible without compromising results. Application of the method to the assessment of the differential proliferative responses of lymphocyte subsets of human peripheral blood mononuclear cells (PBMC) that were costimulated with CD3 and with CD28.2 is presented.