After harvesting and washing, cells were processed by fixation with 75% ethanol overnight. The γH2AX, a phosphorylated form of H2AX, was recognized by p-Histone H2A.X primary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) (50× dilution, 1 h, 4 °C). Subsequently, the secondary antibody-modified with Alexa Fluor®488 (Cell Signaling Technology) (10,000× dilution, 30 min, RT) was used. Finally, cells were stained by 7AAD (final concentration 1 μg/mL, 30 min). Cells were resuspended in PBS. The Accuri C6 flow cytometer detected both γH2AX and 7AAD intensities (FL1 and FL3 channels). γH2AX (+)/7ADD (+) populations were calculated for γH2AX analysis.
The protocol for observing γH2AX foci was slightly modified [41 (link)]. In brief, cells were washed with PBS, fixed with 4% paraformaldehyde (Sigma-Aldrich) for 5 min, permeabilized with 0.5% Triton X-100/PBS for 5 min, and blocked by 1% bovine serum albumin (BSA) for 1 h. Subsequently, cells were incubated with the primary antibody for γH2AX (Santa Cruz, CA, USA) (1:400) for 1 h. After washing, cells were incubated with a secondary antibody with Alexa Fluor 488 label (1:500 dilution) and counterstained with Hoechst 33342 (Sigma-Aldrich) (1:1000 dilution) for 1 h before mounting. Images were captured by a DMi8 microscope (Leica Microsystems, Wetzlar, Germany).
The protocol for observing γH2AX foci was slightly modified [41 (link)]. In brief, cells were washed with PBS, fixed with 4% paraformaldehyde (Sigma-Aldrich) for 5 min, permeabilized with 0.5% Triton X-100/PBS for 5 min, and blocked by 1% bovine serum albumin (BSA) for 1 h. Subsequently, cells were incubated with the primary antibody for γH2AX (Santa Cruz, CA, USA) (1:400) for 1 h. After washing, cells were incubated with a secondary antibody with Alexa Fluor 488 label (1:500 dilution) and counterstained with Hoechst 33342 (Sigma-Aldrich) (1:1000 dilution) for 1 h before mounting. Images were captured by a DMi8 microscope (Leica Microsystems, Wetzlar, Germany).