Quantifying DNA Damage via γH2AX Staining

DNA double-stranded breaks (DSBs) and blocked replication forks lead to the phosphorylation of histone protein H2AX on serine 139 (Muslimovic et al. 2008 (link)). We performed a γH2AX staining to investigate the DNA damage by the procedure as previously described by Mariotti et al. (2013 (link)) with some modifications. Briefly, after treatment with various pH and B[a]P at different time points, A549 cells were fixed in 2% formaldehyde for 20 min at room temperature. After fixation cells were permeabilized with 0.5% Triton X-100:PBS for 20 min at 4 °C and then blocked with 2% BSA and 0.5% Tween-20 in PBS. Cells were then stained with anti-γH2AX antibody (Upstate via Merck Millipore) overnight at 4 °C and the use anti-mouse Alexa fluor 488 secondary antibody (Life Technologies) for 1 h at 37 °C. Coverslips were mounted with VECTASHIELD^® Mounting Medium containing DAPI, to counterstain cellular nuclei. γH2AX stained cells were scored manually by a digital fluorescent microscope with 100X objective, and the average number of positive cells was calculated from a minimum of 100 cells per dose/time point. Experimental data present the average of 4 independent experiments.