Anti h2ax ps139 antibody

Irradiated and nonirradiated PC9 and A549 cells (5 × 10⁴) were cultured with cisplatin and/or bortezomib or with DMSO for 72 h for cell cycle analysis or 48 h for DNA damage analysis. For cell cycle analysis, after incubation for the abovementioned durations, cells were harvested, washed in ice-cold PBS, collected by centrifugation, and fixed with 70% ethanol. DNA was stained with PBS containing PI (50 µg/ml) and RNase A (1 mg/ml) (Sigma). After incubation for 30 min at 4 °C in the dark, the cell cycle distribution was determined using the BD FACS Canto II. To assess DNA damage, after incubation for the abovementioned durations, cells were harvested and labeled with an anti-H2AX (pS139) antibody (BD Pharmingen) for 30 min at 4 °C and analyzed using the BD FACS Canto II. The mean fluorescence index was calculated using FlowJo software (TreeStar, Ashland, USA).

Irradiated and nonirradiated PC9 and A549 cells were cultured with different concentrations of cisplatin and bortezomib for 72 h for cell cycle analysis or 48 h for DNA damage analysis. Apoptotic cell death was assessed by double staining with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI; Apoptosis Detection Kit II, BD Pharmingen) as previously described^{22 (link)}. DNA damage was evaluated by staining cells with an anti-H2AX (pS139) antibody (BD Pharmingen). Fluorescence-activated cell sorting (FACS) data were acquired using a BD FACS Canto II (BD Biosciences, San Jose, CA). The mean fluorescence index was calculated using FlowJo software (TreeStar, Ashland, USA). Gating was implemented based on negative control staining profiles.