Quantifying DNA Damage with γH2A.X Immunostaining

Immunofluorescence-staining of γH2A.X was performed as described before [24 (link)]. Briefly, cells were seeded on cover glasses in 24-well plates, cultured for 24–48 h, and then treated with cisplatin alone or together with Olaparib or ZC-22 for 24 h. After washing with PBS, cells were fixed by 4% PFA, permeated with 0.5% Triton-X100 in PBS, washed thrice with 0.1% PBS-Tween (PBST), blocked with 3% BSA in PBST, and then incubated with anti-γH2A.X antibody overnight at 4 °C. Cells were then washed thrice with 0.1% PBST, incubated with Alexa Fluor 488 conjugated secondary antibody (Cell Signaling, Danvers, MA, USA) for 1 h at room temperature in dark, and mounted with Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were obtained with the Olympus FV1000 laser scanning confocal microscope. At least 150 cells in three fields were counted for each repeat.