Quantification of γH2AX using flow cytometry

We are interested in the induction and repair of DNA double strand breaks (dsb) after ionizing radiation using γH2AX as a marker. In addition to the widely applied fluorescence microscopy of immunostained γH2AX foci, we established the flow cytometric detection of γH2AX in our lab. The adaptation of the staining procedure with regard to trypsinization and centrifugation steps was described previously [1]. Here we set out to check the reproducibility of the results obtained by flow cytometry. For this purpose we first irradiated V79 cells with x-rays in four-fold determinations. Secondly we repeated the flow cytometer measurement of the samples after different times. Figure 1 shows the results for V79 cells stained for γH2AX 30 min after x-ray irradiation. The mean fluorescence of gH2AX was obtained by G0/G1 cell cycle phase cells. The square points demonstrate the measurement directly after finishing the staining procedure (fresh) and the circles show the remeasurement on the next day.