Simultaneous analysis of radio-induced membrane alteration and cell viability by flow cytometry
Benderitter M, Vincent-Genod L, Berroud A, Voisin P,
Cytometry 2000, 39 (2), 151-157
Background : Modifications of intracellular transfer, resulting from a loss of membrane integrity may contribute toward setting the cell onto the pathway of apoptosis. Methods : We have developed an original technique of measuring simultaneously, 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 membrane alterations are linked. Investigations were performed on lymphocytes 24h after whole human blood Y-irradiation. Results : our results confirmed the expected increase in the percentage of apoptotic cell as a function of dose, but revealed that the percentage of necrotic cells appeared stable after irradiation. At the same time, the fluorescence anisotropy of the living lymphocyte subpopulation decreased significantly and dose dependently as measured 24h post-irradiation. With TMA-DPH, the anisotropy index of apoptotic lymphocytes was always lower than that of the viable lymphocyte subpopulation. On the other hand, 1,6-diphenyl-1,3,5-hexatriene (DPH) anisotropy was similar in apoptotic and viable cells after irradiation. These fondings suggest that apoptotic lymphocytes are characterised by a membrane fluidisation that mainly occurs on the cell membrane surface. Conclusion : Our study made technical advances in using cytometric fluorescence anisotropy measurement as an early biological indicator of apoptosis after cellular exposure to ionising radiation.