Apo brdu red in situ dna fragmentation assay kit

To evaluate the hepatocytes' apoptosis after H/R exposure, commercial Apo-BrdU-Red In Situ DNA Fragmentation Assay Kit (BioVision, Cat. # K404-60) was deployed. The Br-dUTP in this kit could actively bind DNA strand breaks, then be identified by a red fluorescence labeled anti-BrdU monoclonal antibody, and ultimately be read by FCM (Ex/Em: 488/576 nm). In brief, L02 hepatocytes after H/R treatment were collected, resuspended into PBS containing 1% (w/v) formaldehyde, and stored in 4°C. Further procedures were performed according to the instructions contained in the kit.

DNA fragmentation due to shRNA treatment was studied using Apo-BrdU-Red In-situ DNA fragmentation assay kit (Biovision, California, USA). HSP70-2 shRNA3, shRNA4 and NC shRNA transfected MDA-MB-231 cells were processed as described earlier [13 (link)]. The cells were analyzed at 576 nm using BD-FACS VERSA. (BD Biosciences, California, USA).

Cell cycle arrest after shRNA treatment was evaluated by PI staining as described earlier [16 ]. Briefly, after 48 hr of shRNA treatment cells were washed and fixed in 70% ethanol overnight. Next day cells were treated with PI stain (1 mg/ml) and RNAse solution and kept in dark at 37°C for 30 min before acquisition. Acquisition and analysis was done using BD-FACS CALIBUR (BD Biosciences, California, USA) and BD FACS Verse BD FACSuite software (BD Biosciences San Jose USA). Caspase mediated DNA nicking was analyzed using Apo-BrdU- Red in-situ DNA fragmentation assay kit (Biovision, Milpitas, CA, USA) as described earlier [16 ]. Acquisition and analysis was done using BD FACS Verse BD FACSuite software (BD Biosciences San Jose USA).