Flow Cytometric Analysis of Phosphorylated Histone H2AX Following Exposure to Ionizing Radiation in Human Microvascular Fndothelial Cells

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We applied a flow cytometric method to quantify IR-induced histone H2AX phosphorylation at serine 139 (γH2AX) and compared those values to those obtained using a standard microscopy based foci counting method. After PFA fixation, methanol permeabilization was suitable for both FITC- or Alexa647-γH2AX. In contrast, Alexa647-γH2AX was not suitable for ethanol permeabilization. Antibody concentrations at 1-2μg/ml yielded the highest γH2AX positive percentage for both antibodies. Without DAPI staining, γH2AX formation can be measured as a relative fold increase. Values determined by bivariant flow cytometric analysis and those obtained using microscopic foci formation exhibited a good quantitative correlation. Values obtained by both methods could vary according to the gating or threshold setting used. γH2AX positive cells increased as a function of radiation dose (2-16 Gy) followed by a dosedependent decay. The free radical scavenger N-acetyl-L-cysteine (NAC), if administered at a concentration of 4mM 30min before IR, was effective in reducing IR-induced γH2AX formation in all phases of the cell cycle. We have developed a simplified and quantitative flow cytometry based method to measure IR-induced γH2AX in cells and demonstrated strong correlation to values obtained by a standard automated digital microscopic foci analysis along with NIH ImageJ custom macro software (Appendix).