Elevated H2AX Phosphorylation Observed with kINPen Plasma Treatment Is Not Caused by ROS-Mediated DNA Damage but Is the Consequence of Apoptosis

Figure 2

Analysis of γH2AX in TK6 cells and its relation to ROS. (a–c) Gating strategy of TK6 cells at 2 h after treatment with agents was done by first including cells in time (a) and forward (FS) and side scatter (SS) cell gate (b), before excluding doublets and subG1 cells for the singles gate (c). (d–e) Singles were then analyzed for total γH2AX as exemplified with representative fluorescence histogram overlay (d), subjected to algorithm-driven cell cycle analysis (e), or manually gated for each cell cycle phase (f) and subsequent determination of γH2AX^hi cells in histograms (g). (h) Confirmation of γH2AX foci (green) in DAPI-stained nuclei (blue) by confocal laser scanning microscopy. (i) Quantification of total (independent of cell cycle phase) γH2AX with treatments and presence or absence of antioxidants. (j) Quantification of total γH2AX within each cell cycle phase at 2 h after treatment with agents in the presence or absence of antioxidants. Quantification (i, j) was done by multiplying the percent of cells positive for γH2AX (% gated) with the mean fluorescent intensity (MFI) of γH2AX⁺ cells. Data show violin plots (i) or single values and (j) of three independent experiments with duplicates each. Statistical analysis (i) within each treatment group was done with one-way ANOVA and Dunnett post hoc test to vehicle control. Scale bar (h) is 10 μm; ns = not significant.