γ h2ax primary antibody

Immunofluorescence assay was used to detect the number of γ-H2AX foci. Cells were seeded in 24-well plates. After washing with PBS, cells were fixed in 3% paraformaldehyde and permeabilized in 0.1% Triton X-100 in PBS. The cells were then stained with γ-H2AX primary antibody (Cell Signaling Technology; 1:200) and thereafter the secondary antibody (fluorescein goat anti-rabbit IgG, Invitrogen; 1:1000). Cells were stained with a DAPI (Antifade Mounting Medium with DAPI, Beyotime). The images were captured using an Olympus BX60 fluorescent microscope (Olympus America Inc).

U251 and GSC11 cells were seeded on coverslips with 50 to 70% confluence and were then washed with cold PBS, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.5% Triton X-100 solution for 10 min on ice. Samples were blocked with 3% bovine serum albumin in PBS for 30 min, incubated with the γ-H2AX primary antibody (Cell Signaling Technology, #9718) overnight at 4°C, and washed three times with PBS for 5 min each. They were then incubated with Alexa Fluor 488–conjugated goat anti-rabbit immunoglobulin G (IgG) (H+L) highly cross-adsorbed secondary antibody (Invitrogen, #A32731) for 2 hours at room temperature in the dark, followed by rinsing three times with PBS for 5 min each. Coverslips were mounted on the microscope slides with Vectashield Mounting Medium containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, #H-1200-10) and analyzed using a Leica SP8 confocal microscope to quantitate the number of nuclei positive for foci at the Advanced Microscopy Core Facility of MDACC. The cells with more than five foci in the nucleus were considered as γ-H2AX foci–positive ones.

Following knockdown and/or treatments, cells were plated at a density of 15,000 cells/well in wells of Lab-Tek chamber slides (Sigma-Aldrich, St. Louis, MO, USA) and fixed in pre-chilled methanol for 5 minutes at −20 °C. Cells were washed with PBS and incubated with blocking buffer (5% BSA/PBS) at room temperature for one hour. The cells were then incubated at 4 °C with γH2AX primary antibody (#2577 S, Cell signaling, Danvers, MA, USA) diluted 1:400 in blocking buffer on a shaker overnight. Following primary antibody incubation and PBS washes, cells were incubated with Alexa-fluor-647 secondary antibody (Thermofisher, Waltham, MA, USA) in blocking buffer at room temperature for an hour. For ABCB1 staining, an ABCB1/MDR-1 mouse primary (D-11, Santa Cruz Biotechnology, Dallas, TX, USA) and a Cy5 affinipure anti-mouse secondary (Jackson Immunoresearch Labs, West Grove, PA, USA) were used. The cells were then washed again with PBS and incubated with a DAPI stain solution (1 µg/ml in PBS) for 5 minutes at room temperature. After additional washing steps, cells were imaged on a Leica DMi8 Microscope (Leica, Wetzlar, Germany). γH2AX foci were analyzed and quantified using ImageJ (NIH, Bethesda, MD, USA). For Doxorubicin efflux experiments, mean fluorescence intensity was computed using NIS-Elements (Nikon Instruments, Melville, NY, USA).