Detection of Apoptosis in Live Cells by MitoTracker Red CMXRos and SYTO Dye Flow Cytometry

We characterized the ability of six SYTO nucleic acid stains and a mitochondrial stain to resolve by flow cytometry camptothecin-induced apoptotic and non-apoptotic cells. Staining live human lymphoid B-cells showed such resolution with SYTO 11, 12, 13, 14, and 16 dyes. H9, HL-60, and Jurkat cells did not show resolution with the SYTO 12 dye, but did with the others. SYTO 15 dye did not show resolution with any cell type. RNase A treatment of fixed lymphoid B-cells strongly reduced fluorescence after staining with SYTO 12 dye; the other SYTO dyes showed little or no RNase A sensitivity. Reduced SYTO 12 fluorescence may reflect RNA breakdown during apoptosis, while decreased fluorescence of the other SYTO dyes in apoptotic cells may be due to chromosomal alterations during apoptosis. In all cell types tested, clear resolution between apoptotic and non-apoptotic cells was observed with the MitoTracker Red dye CMXRos. In double-staining experiments, cells exhibiting reduced SYTO 11 fluorescence were the same as those showing decreased CMXRos fluorescence. We conclude that changes in nucleic acid stability or conformation may vary during apoptosis from one cell type to another, but mitochondrial demise may be common to all apoptotic pathways.