Combined hoechst 33342 and merocyanine 540 staining to examine murine B cell cycle stage, viability and apoptosis

A procedure is described for the dual staining of lymphocytes with Hoechst 33342 (Ho342) to examine cell cycle position, and merocyanine 540 (MC540) that allows for the analysis of cells entering the early stages of apoptosis. Ho342 is a DNA specific dye and MC540 detects membrane phospholipid domain changes, some of which are associated with apoptotic cells. Flow analysis of B cells dually stained with Ho342 and MC540 allows for the discrimination of five distinct subpopulations. Two of these subpopulations represent viable, MC540 negative/dull cells with either 2n or 4n DNA. As 2n and 4n DNA B cells become MC540 bright they move into two distinct subpopulations representing cells entering and progressing through the early stages of apoptosis. As the apoptotic, MC540 bright cells move into the latter stages of apoptosis, they localize into a fifth subpopulation displaying reduced staining with Ho342 indicative of late stage apoptotic cells in the process of fragmenting their DNA. This experimental approach enables the characterization of lymphocyte populations for percentages of viable, early apoptotic, and late apoptotic cells. The cells are not fixed during this procedure, and since both dyes are viable dyes there is an additional opportunity to obtain sorted cells from any of the defined subpopulations for reculturing and functional analysis.