Rabbit monoclonal anti γh2a x antibody

After treated for 48 h, 2 x 10⁵ NALM-6/R cells were subjected to apoptosis assay using an Annexin V Apoptosis Detection Kit-APC (eBioscience Company, USA), following the manufacturer's instructions. For the mitochondrial membrane potential assessment, after different treatments for 48 h, 2 x 10⁵ NALM-6/R cells were analyzed using a JC-1 fluorescent probe kit (Beyotime Company, China), following the manufacturer's instructions. To measure the intracellular reactive oxygen species (ROS) levels, about 2 x 10⁵ NALM/R cells subjected to different treatments were washed in PBS buffer twice, and then incubated in 1 ml of serum-free RPMI 1640 medium containing 10 μM of H₂DCFDA for 30 min at 37°C. The cells were harvested, and then washed in serum-free RPMI 1640 medium buffer twice to remove the remaining H₂DCFDA. The fluorescent intensity was measured by flow cytometry. For assessing the degree of DNA damage, 2 x 10⁵ NALM-6/R cells were incubated for 15 min on ice in hybridization buffer (PBS containing 0.5% bovine serum albumin (BSA) and 0.25% Triton X-100). After centrifugation, the cells were incubated with rabbit monoclonal anti-γH2A.X antibody (Cell Signaling Technology, USA) for 1 h, then washed with PBS and incubated with an FITC-conjugated mouse anti-rabbit IgG antibody (BD Pharmingen) for 30 min in the dark at room temperature.

Rabbit polyclonal anti-CK2 antibody, rabbit polyclonal anti-Gapdh antibody, and rabbit monoclonal anti-γH2A.X antibody were purchased from Cell Signaling Technology (Danvers, MA, USA); Mouse monoclonal anti-α-tubulin-FITC antibody and anti-acetyl-α-tubulin (K40) antibody were purchased from Sigma (St. Louis, MO, USA); Rabbit polyclonal anti-phosphoCK2 and sheep polyclonal anti-BubR1 antibodies were obtained from Abcam (Cambridge, MA, USA); goat anti-mouse IgG (H + L)-FITC, goat anti-rabbit IgG (H + L)-FITC and donkey anti-sheep IgG (H + L)-FITC were obtained from Zhongshan Golden Bridge Biotechnology (Beijing, China).

Cells were plated in 30-mm dishes and cultured for 72 h at 37°C. To detect irradiation-induced DNA double-strand breaks (DSBs), cells were treated with a 2-Gy dose of irradiation from an external X-ray source (RAD SOURCE) at room temperature and incubated for 0.5 and 24 h. Unirradiated cells served as controls. To detect H2AX phosphorylation, cells were sequentially fixed in 4% formaldehyde (Sigma–Aldrich) for 15 min and 50% methanol in PBS for 10 min. The cells were subsequently blocked with 5% bovine serum albumin for 30 min, incubated with a rabbit monoclonal anti-γH2AX antibody (1:1,000, Cell Signaling Technology, Boston, USA) for 30 min, washed in PBS, incubated with an Alexa 488-conjugated (Molecular Probes, USA) secondary antibody for 30 min, and counterstained with DAPI (Invitrogen). Images were captured using an Olympus FV100 confocal microscope. γH2AX-positive cells were defined as those with more than 20 γH2AX foci. Five random fields per coverslip were selected to calculate the number of γH2AX-positive cells. Assays were performed in triplicate to eliminate intra-assay variability.