A new flow cytometric method for discrimination of apoptotic cells and detection of their cell cycle specificity through staining of F-actin and DNA
Article first published online: 8 MAR 2005
Copyright © 1995 Wiley-Liss, Inc.
Volume 20, Issue 2, pages 162–171, 1 June 1995
How to Cite
Endresen, P. C., Prytz, P. S. and Aarbakke, J. (1995), A new flow cytometric method for discrimination of apoptotic cells and detection of their cell cycle specificity through staining of F-actin and DNA. Cytometry, 20: 162–171. doi: 10.1002/cyto.990200209
- Issue published online: 23 JUN 2005
- Article first published online: 8 MAR 2005
- Manuscript Accepted: 15 FEB 1995
- Manuscript Received: 22 NOV 1994
- flow cytometry;
- propidium iodide;
- cell cycle
Drug-initiated apoptosis of human leukemia HL-60, THP-1, and U-937 cells was studied via multiparameter flow cytometry and cell sorting. A new flow cytometric method that allows both identification and quantitation of apoptotic cells and estimation of their cell cycle specificity is presented. The method is based on paraformaldehyde fixation followed by staining of F-actin and DNA with fluorescein isothiocyanate (FITC)-phalloidin and propidium iodide (PI), respectively. Bivariate green fluorescence (F-actin) vs. side scatterplots of HL-60 cells treated with 10 μM etoposide for 4 h showed two cell populations, one with high green fluorescence and low side scatter and one with low green fluorescence and high side scatter. Sorting revealed cells with intact nuclei in the high green fluorescence/low side scatter population and cells with fragmented nuclei in the low green fluorescence/high side scatter population, demonstrating that the cells in the latter population were apoptotic. Exposure of HL-60 cells to 10 μJM etoposide for 4 h resulted in S-phase selective apoptosis, whereas 5 μg/ml cycloheximide initiated apoptosis mainly in G0/G1-phase and S-phase cells. The apoptotic response of HL-60 cells to 20 GY γ-irradiation was selective for S-phase and G2 + M-phase cells. The present method offers the opportunity to estimate the cell cycle distributions of both the apoptotic and the nonapoptotic cell populations, which is especially valuable when apoptosis occurs in association with cell cycle perturbations. A similar shift from one to two cell populations in green fluorescence vs. side scatterplots, similar to that observed for HL-60 cells, was observed in the THP-1 and U-937 cell lines secondary to etoposide treatment. © 1995 Wiley-Liss, Inc.