8 ohdg fitc antibody

DNA damage was detected by probing with the markers γH2AX [33 (link)] and 8-OHdG [34 (link)]. Briefly, cells were fixed overnight. After washing with PBS, γH2AX was detected by γH2AX antibody [33 (link)] (Santa Cruz Biotechnology; Santa Cruz, CA, USA) (4 °C, 1 h) and Alexa Fluor^®488-labeled secondary antibody (Cell Signaling Technology). Subsequently, 7AAD (5 μg/mL, 30 min) was added. 8-OHdG was detected by 8-OHdG-FITC antibody (Santa Cruz Biotechnology) (4 °C, 1 h). The γH2AX and 8-OHdG intensities were examined by flow cytometry (Accuri C6).

Following the cell harvesting and PBS washing, the oxidative DNA damage (8-OHdG) was determined by antibody-associated flow cytometry [35 (link)]. Generally, the fixed cells (Ca9-22 and CAL 27) were mixed with 8-OHdG-FITC antibody (1:10,000 dilution) (Santa Cruz Biotechnology) (4 °C, 1 h) for the flow cytometry and FlowJo analysis. The cells were resuspended in PBS before the flow cytometry.

Utilizing flow cytometry, 8-OHdG, an oxidative nucleotide marker, was assessed using an 8-OHdG-FITC antibody (Santa Cruz Biotechnology) at a 100X dilution for 1 h at RT [55 (link)]. Finally, flow cytometry analysis was performed using an Accuri C6 using the FL1 channel.

Cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. The cells were incubated for 10 min with PBS containing 0.25% Triton X-100 at room temperature. After cells were washed in PBS three times for 5 min, the cells were incubated in 5% BSA in PBS for 1 h at room temperature. Cells were then incubated with 8-OHdG-FITC antibody at 1:50 dilution (sc-393871, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in 5% BSA in PBS overnight at 4°C before examination using a fluorescence microscope. For caspase blocking, cells were pretreated with caspase-9 inhibitor (BD bioscience 550381, Z-LEHD-FMK, BD Biosciences, San Jose, CA, USA) or caspase-8 inhibitor (BD bioscience 550380, Z-IEDH-FMK) for 2 hours before treatment with SR-T100.

Both γH2AX and 8-hydroxy-2-deoxyguanosine (8-OHdG) were chosen as cellular DNA damage markers. Following fixation, the γH2AX [39 (link)] and 8-OHdG [40 (link)] expressions were detected by antibody-based methods. For γH2AX measurement, Santa Cruz Biotechnology γH2AX antibody (Santa Cruz, CA, USA) (4 °C, 1 h) and Alexa Fluor^®488-linked secondary antibody (Cell Signaling Technology) were sequentially applied to fixed cells, and counterstained with 7AAD (5 μg/mL, 30 min). For 8-OHdG measurement, the 8-OHdG-FITC antibody (Santa Cruz Biotechnology) (4 °C, 1 h) was applied. In addition, their intensities were monitored by Accuri C6 flow cytometry.

DNA damage was detected with γH2AX [29 (link)] and 8-hydroxy-2-deoxyguanosine (8-OHdG) [9 (link)]. The γH2AX antibody [30 (link)] was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Subsequently, Alexa Fluor^®488-secondary antibody (Cell Signaling Technology) and 7AAD were added for double staining. Additionally, 8-OHdG was examined with the 8-OHdG-FITC antibody (Santa Cruz Biotechnology). Finally, the as-prepared cells were subjected to flow cytometry.

γH2AX [14 (link)] and 8-OHdG [40 (link)] were detected through their targeted antibodies, and their levels were assessed by the flow cytometer as described. The primary antibodies (4 °C, 1 h) of γH2AX and the 8-OHdG-FITC antibody (Santa Cruz Biotechnology; Santa Cruz, CA, USA) were applied to 75% ethanol-fixed cells. 7AAD (5 μg/mL, 30 min) was further added for γH2AX detection.