4.9. Determination of γ-H2AX Formation

For γ-H2AX foci visualization, cells grown on Lab-Tek chambers (Nunc, Thermofisher, Illkirch, France) were treated as indicated earlier and fixed with a 4% paraformaldehyde solution for 15 min. Then samples were permeabilized with PBS containing 0.5% Triton-X-100 and blocked with 10% BSA. Then, cells were incubated overnight at 4 °C, with blocking buffer containing primary antibody against γ-H2AX (clone JBW301; Merck Millipore, Darmstadt, Germany) followed by incubation with Alexa Fluor 488 goat anti-mouse IgG (Invitrogen) for 3 h at room temperature. Samples were counterstained with antifading agent containing DAPI. The formation of foci in nuclei were monitored by immunofluorescence using an Olympus BH-2 fluorescence microscope equipped with digital camera.

For γ-H2AX analysis in flow cytometry, 24 h after treatment, cells were harvested and washed in ice-cold PBS, fixed in 1 mL of 70% ethanol and stored at −20 °C for 24 h. Fixed cells were centrifuged and pellet was resuspended twice in 2 mL cold PBS containing 10% bovine serum albumin (BSA, Sigma-Aldrich, Lyon, France) and 0,2% Triton-X-100 (T-PBS-BSA). Then, labelling was performed using a solution of monoclonal mouse anti-phospho-histone-H2AX (ser 139) polyclonal antibody (clone JBW301; Merck Millipore, Darmstadt, Germany) diluted in 1/100 in T-PBS-BSA. Cells were stored at 4 °C overnight. Cells were washed twice with 2 mL T-PBS-BSA and resuspended in 100 µL of a solution containing an anti-mouse IgG fluorescein-conjugated antibody diluted at 1:200 (Invitrogen) and were stored 1 h in room temperature and gently shaken. Then, 2 mL T-PBS-BSA wad added, cells were centrifuged and pellet was resuspended in 200 µL of PBS containing PI (5 µg/mL) and RNAse A (100 µg/mL). Next, cells were stored at room temperature in the dark for 30 min and gently shaken. The fluorescence of 10,000 cells was analyzed using a BD Accuri^TM C6 flow cytometer and software (BD). For quantification of γ-H2AX positivity, a gate was arbitrarily set on the control, untreated sample to define a region of positive staining for γ-H2AX of approximately 5% [22]. This gate was then overlaid on the treated samples.