Simultaneous analysis of radio-induced membrane alteration and cell viability by flow cytometry

Background: Modifications of intracellular transfer, resulting from a loss of membrane integrity may contribute toward setting the cell onto the pathw ay of apoptosis. Methods: We have developed an original technique of measuring simultaneousl y, with flow cytometry, changes in membrane fluidity and cell death status. Our aim was to assess the extent to which radio-induced cell death and mem brane alterations are linked. Investigations were performed on lymphocytes 24 h after whole human blood gamma-irradiation. Results: Our results confirmed the expected increase in the percentage of a poptotic cells as a function of dose, but re revealed that the percentage o f necrotic cells appeared stable after irradiation. At the same time, the f luorescence anisotropy of the living lymphocyte subpopulation decreased sig nificantly and dose dependently as measured 24 h post-irradiation. With TMA -DPH, the anisotropy index of apoptotic lymphocytes was always lower than t hat of the viable lymphocyte subpopulation. On the other hand, 1,6-diphenyl -1,3,5-hexatriene (DPH) anisotropy was similar in apoptotic and viable cell s after irradiation. These findings suggest that apoptotic lymphocytes are characterised by a membrane fluidisation that mainly occurs on the cell mem brane surface. Conclusion: Our study made technical advances in using cytometric fluoresce nce anisotropy measurement as an early biological indicator of apoptosis af ter cellular exposure to ionising radiation. Cytometry 39:151-157, 2000. (C ) 2000 Wiley-Liss, Inc.