Antibody against γh2ax

Cells grown on coverslips were fixed with 4% paraformaldehyde for 15 min at room temperature and permeabilized with 0.5% Triton X1−00 for 10 min. Cells were further incubated with ice-cold methanol for 5 min and blocked with 3% BSA in PBS for 30 min. Coverslips were incubated with primary antibodies for 3 h, washed with PBS, and incubated with AlexaFluor-conjugated secondary antibodies for 1 h. Mounting was performed using Vectashield. Imaging was performed using Leica Sp8 confocal microscope equipped with 63× oil objective (NA 1.40). Images were analyzed using LAS AF Lite software (Leica, Wetzlar, Germany). Induction of DNA damage response was evaluated as described previously [32 (link)]. Briefly, cells were exposed to ionizing radiation (3 Gy) using X-RAD 225XL instrument (Precision; Cu filter 0.5 mm), fixed with 4% PFA, permeabilized with 0.5% Triton X1−00, and probed with antibody against γH2AX (Cell Signaling Technology). Images were acquired using Olympus ScanR system equipped with 40×/NA 1.3 objective (Olympus, Tokio, JApan). Number of γH2AX-positive foci per nucleus was determined using spot detection module. More than 300 nuclei were quantified per condition.

Human ovarian cancer cells A2780 and A2780/DDP were obtained from the American Type Culture Collection (ATCC) and PEO14 was kindly provided by Dr. Charlie Gourley (Edinburgh, UK). Both ovarian cancer cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) complete medium, supplemented with 10% fetal bovine serum (FBS). Normal ovarian epithelial cell line IOSE80 was obtained from Canadian Ovarian Tissue Bank and grown in medium 199 (Invitrogen) and MCDB 105 (Sigma), supplemented with 10% FBS. Rabbit anti-human ERCC1 and β-actin polyclonal antibodies were purchased from Proteintech Group lnc. (Chicago, USA). Cisplatin was purchased from Sigma (St. Louis, MO, USA) and MEK inhibitor PD0325901 was obtained from Selleck (St. Louis, MO, USA). Antibody against γH2AX was purchased from Cell Signaling Technology (MA, USA). Antibody against ERCC1 was purchased from Santa Cruz (CA, USA).