Flow cytometric analysis of apoptotic subpopulations with a combination of Annexin V-FITC, propidium iodide, and SYTO 17

BACKGROUND: We have previously characterized apoptotic cell death induced in a follicular lymphoma cell line, HF-1, after triggering via the B-cell receptor (BCR) or treatment with Ca(2+) Ionophore A23187. We analyzed the kinetics of apoptosis induced by these two treatments, as two alternative models of classical apoptosis, by flow cytometry using a novel combination of cytofluorometric stains. METHODS: Cells were stained with a combination of Annexin V-FITC, propidium iodide (PI), and SYTO 17 and analyzed by a two-laser flow cytometry system using 488-nm argon and 633-nm HeNe air-cooled lasers. RESULTS: In both apoptotic models, the first apoptotic cells were detected by SYTO 17 staining. The alteration in SYTO 17 staining intensity was followed by an increased uptake of PI. Finally, the apoptotic cells were labeled with Annexin V in BCR-induced apoptosis. On the contrary, on treatment with Ca(2+) Ionophore A23187, cells became positive for Annexin V earlier than for PI. CONCLUSIONS: The novel cytofluorometric dye, SYTO 17, discriminates apoptotic alterations before Annexin V and PI. PI also discriminates apoptotic alterations before the loss of plasma membrane asymmetry by BCR but not by Ca(2+) Ionophore A23187-induced apoptosis. Finally, the combination of these three cytofluorometric dyes allows effective detection of apoptotic subpopulations and ordering of apoptotic events by flow cytometry.