Mouse monoclonal antibody against human γ h2ax

Clarithromycin (CAM) was purchased from Selleck Chemicals (Houston, TX, USA); it was dissolved in dimethyl sulfoxide (DMSO). DDP was obtained from Sigma-Aldrich (St. Louis, MO, USA); it was dissolved in sterile filtered RNA-free water. N-acetyl cysteine (NAC) was purchased from Beyotime Biotechnology (Shanghai, China). Antibodies used for analysis included the following: primary rabbit polyclonal antibody against human cleaved-PARP (Epitomics, Burlingame, CA, USA), rabbit monoclonal antibody against human activated caspase-3 (Cell Signaling Technology, Inc., Beverly, MA, USA), rabbit monoclonal antibody against human SOD2 (Proteintech, Wuhan, China), goat monoclonal antibody against human 8-OHdG (Millipore, Bedford, MA, USA), and mouse monoclonal antibody against human γ-H2AX (Millipore, Bedford, MA, USA). The secondary antibody for GAPDH was obtained from Santa Cruze Biotech (Santa Cruz, CA, USA).

After treatment as described above, H2AX phosphorylation was analyzed in cells derived from monolayer cultures following incubation with trypsin-EDTA. Cells were pelleted by centrifugation, then fixed in 70 % ethanol and stored at −20 °C. Fixed cells were washed one time with phosphate-buffered saline (PBS) containing 1 % bovine serum albumin (BSA) and blocked with 10 % horse serum and 0.5 % Tween20 in PBS at room temperature for 15 min. Cells were incubated with a mouse monoclonal antibody against human γH2AX (1:300; Millipore, Billerica, MA) in PBS with 1 % BSA and 0.5 % Tween20 at room temperature for 1 h. After one wash with PBS with 1 % BSA, cells were incubated with an Alexa Fluor 488-conjugated goat anti-mouse IgG antibody (1:400; Molecular Probes) at room temperature for 1 h. Cells were washed two times and subsequently permeabilized with 400-μl PBS containing 0.1-mg/ml PI, 0.1 % Triton X-100, and 400-μl (0.8 mg) DNase-free RNaseA at room temperature in the dark for 30 min, then kept on ice. Samples were measured using a FACSCalibur (BD, Franklin Lakes, NJ). Data were analyzed with FlowJo software (Digital Biology, Tokyo, Japan).