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Simultaneous analysis of radio-induced membrane alteration and cell viability by flow cytometry
M Benderitter1, L Vincent-Genod, A Berroud
1Institut de Protection et de Sûreté Nucléaire, IPSN, Fontenay-aux-Roses, France. marc.benderitter@ipsn.fr
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 24 h after whole human blood gamma-irradiation.
Results:
Our results confirmed the expected increase in the percentage of apoptotic cells 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 24 h 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 findings suggest that apoptotic lymphocytes are characterised by a membrane fluidization 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.