P histone h2ax antibody

p-Histone H2AX antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to detect γH2AX. Subsequently, fixed cells were incubated with Alexa Fluor^® 488-secondary antibody (Cell Signaling Technology, Danvers, MA, USA) [33 (link),34 (link)], accompanied by 7AAD (1 μg/mL, 30 min) incubation. 8-OHdG-FITC antibody (1:10,000 dilution) (Santa Cruz Biotechnology) was used to detect 8-OHdG [35 ]. Finally, cells were resuspended in 1× PBS for flow cytometry.

Since γH2AX is a protein for targeting DNA double-strand breaks, monitoring the relationship of γH2AX and cellular DNA is reasonable. For the γH2AX assay, p-Histone H2A.X antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used to recognize the phosphorylated H2AX (γH2AX). Then, the Alexa Fluor^®488-secondary antibody (Cell Signaling Technology, Danvers, MA, USA) was applied [62 (link)]. 7AAD (1 μg/mL, 30 min) was further applied to stain cellular DNA. Both γH2AX and 7AAD (+) populations were examined for DNA damage with double-strand breaks. The Accuri C6 flow cytometer detected the Alexa Fluor^®488 intensities for γH2AX contents.