Mouse anti γh2ax antibody

A DSB marker, γ-H2AX foci formation assay was carried out as it was previously (23 (link)). Briefly, each slide containing cells was taken out of the flask following irradiation and was washed in cold PBS and fixed for 15 min in 4% w/v paraformaldehyde in PBS and washed again in PBS. Cells were then permeabilized for 5 min in 0.2% v/v Triton X-100 (Sigma, St Louis MO, USA) in PBS and washed twice in PBS. Slides were treated with 10% goat serum for 1 h at 37°C for blocking. Antibodies were diluted with 10% v/v goat serum in PBS. Cells were incubated with 1:300 diluted mouse anti-γ-H2AX antibody (Millipore, Burlington, MA, USA) for 1 h at 37°C, washed three times in PBS, and incubated with 1:500 diluted Alexa Fluor 488 goat anti-mouse IgG antibody (Abcam, Cambridge, MA, USA) for 1 h at 37°C, and washed four times in PBS. DAPI (4′,6-diamidino-2-phenylindole) (Roche, Indianapolis, IN, USA) in SlowFade was then applied to stain the DNA.

Monoclonal antibodies against Bcl-2, Bax, P53, P21, Mcl-1, and GAPDH were purchased from Santa Cruz (Santa Cruz, CA, USA). Antibodies against CytC, Fas, FasL, and TRAIL were purchased from Abcam (Cambridge, MA, USA). Mouse anti-γ-H2AX antibody was from Millipore (Billerica, MA, USA). DMEM medium and FBS were purchased from Gibco BRL (Grand Island, NY, USA). FITC annexin V Apopotosis Detection Kit was from BD Biosciences (San Jose, CA, USA). Caspase-Glo 3/7, 8, and 9 assay kits and Plasmid extraction kit were purchased from Promega (Madison, WI, USA). N-acetyl-L-cysteine (NAC) and Reactive Oxygen Species Assay Kit were purchased from Sigma-Aldrich (St. Louis, MO, USA). Lipofectamine^TM 2000, Dichloro-dihydro-fluorescein diacetate (DCFH-DA), MitoSOX^TM Red, MitoTracker Deep Red FM, MitoTracker Green FM and Hoechst 33342 were from Invitrogen (Carlsbad, CA, USA). Trypan Blue Staining Cell Viability Assay Kit was from Beyotime Institute of Biotechnology (Jiangsu, China). Tetramethylrhodamine methyl ester (TMRM) was from Life Technologies (Carlsbad, CA, USA). Z-LEHD-FMK, Z-IETD-FMK, Z-DEVD-FMK were from Calbiochem (San Diego, CA, USA). Colorimetric TUNEL Apoptosis Assay Kit was from Beyotime (Shanghai, China). Mitochondria/Cytosol Fractionation Kit was from BioVision (San Francisco, CA, USA). DL-2000 DNA Marker was from TaKaRa Biotechnology (Dalian, China).

At 48 (+2) h postinfection, Salmonella-infected HIEC-6 cells and controls were washed once with PBS and subsequently harvested with 0.25% trypsin-EDTA. The positive-control cells were incubated with H₂O₂ at a final concentration of 200 μM for 1 h at 37°C. Cells were fixed with 4% formaldehyde (Thermo Fisher Scientific) at room temperature for 5 min and permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Cells were then blocked with 3% BSA (Sigma-Aldrich) in PBS for 30 min. Antibody staining was performed at room temperature for 1 h with mouse anti-γH2AX antibody (Millipore) and for 30 min with donkey anti-mouse conjugated to Alexa Fluor 488 (Thermo Fisher Scientific); both antibodies were used at a 1:200 dilution. Labeled cells were subsequently stained for cell cycle analysis by incubation with PI for 10 min at room temperature (using 300 µl of the PI staining solution described above). Cells were analyzed using the BD FACSAria sorter within 2 h of staining. The gating strategy used is shown in Fig. S3.

MSTO-211H cells were plated on slides and treated for 72 h with diluent or 5 μM complex 6. Slides were then fixed for 20 min with 4% paraformaldehyde (Sigma-Aldrich), rinsed with PBS, and then the cells were permeabilized for 5 min in 0.1% Triton X-100, followed by three 5 min PBS washes. Slides were treated thrice for 10 min in a blocking solution of 5% bovine serum albumin (BSA) (Invitrogen). Mouse anti-γH2AX antibody (Millipore) was added (1:100 in 1% BSA) and incubated for 1 h in a dark, humidified environment at room temperature. Slides were then exposed to a secondary goat anti-mouse antibody, FITC (1:100 in PBS) and incubated for 1 h in the dark, humidified environment at room temperature. Slides were rinsed thrice in PBS, mounted, and cell imaged with a Leica fluorescence microscope.

Exponentially growing cells were seeded into chamber slides (BD Falcon, Heidelberg, Germany) at 5×10³ cells per well and treated as described in the results section. At the specified times after irradiation, samples were fixed in 3.7% formaldehyde with 0.2% Triton-X-100, blocked with 1% bovine serum albumin for 30 min and incubated for 1 h with mouse anti-γH2AX antibody (Millipore, Schwalbach, Germany) at room temperature. The cells were washed three times with PBS for 10 min, and then incubated with FITC-conjugated goat-anti-mouse secondary antibody (Millipore) for 1 h, washed and mounted. 50–100 cells were scored for each condition.

Cells were grown on cover slips and treated with RSV or DMSO (0.01%). After treatment, cells were washed in PBS twice and were fixed in 4% paraformaldehyde for 20 min at room temperature, followed by washing in PBS for three times and treatment with 0.2% Triton X-100 in PBS for 15 min. Cells were then further washed in PBS twice and then blocked with 10% normal goat serum in PBS for 60 min, following which mouse anti-γ-H2AX antibody (Millipore, Billerica, MA) was added at a dilution of 1:200 in 5% normal goat serum in PBS and incubated overnight at 4 °C. Next day, cover slips containing cells were then washed and incubated for one hour at 37 °C in the dark with the Rhodamine-labeled secondary antibody at a dilution of 1:200 in 5% normal goat serum in PBS for 60 min. The secondary antibody solution was then aspirated and the cells were washed four times in PBS. Cells were then incubated in the dark with 4-6-diamidino-2-phenylindole (DAPI) for 5 min and coverslips were mounted with an antifade solution (Molecular Probes, Eugene, OR). Slides were then examined under a fluorescent microscope. For each treatment condition, γ-H2AX foci were counted in at least 100 randomly captured cells.

Cells were passaged onto slides, exposed 24 h later to 4 Gy of γ-irradiation, and incubated at 37 °C for 4 h. Cells were washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, washed again with PBS, permeabilized for 10 min using 0.05% Triton X-100 and 0.5% NP-40, and then washed three times (5 min each) in PBS. The cells were blocked for 1 h with 2% bovine serum albumin (BSA), and then incubated for 1 h at room temperature with a mouse anti-γH₂AX antibody (1:500; Millipore, Temecula, CA, USA). Cells were washed three times with PBS containing 0.05% Tween 20, and then incubated with a goat anti-mouse secondary antibody (1:800; Abcam) for 1 h in the dark at room temperature. Cells were counterstained with 0.2 mg/mL 4′,6-diamidino-2-phenylindole (DAPI, 1:2000; Sigma, Shanghai, China). Confocal images were acquired and analyzed using a TCS SP5 (Leica) microscope equipped with an HCX PL 63 × 1.4 CS oil-immersion objective lens.