Apo brdu in situ dna fragmentation assay kit

The apoptosis of cardiomyocytes was examined by TUNEL assay. Briefly, cultured cardiomyocytes were fixed with 4% paraformaldehyde (Millipore, USA) and permeabilized with 1% Triton X-100 (Sigma Aldrich, USA) and in phosphate-buffered saline (PBS) (Invitrogen, USA) for 30 min, followed by 3 times (3 × 10 min) wash of fresh PBS. Then, an Apo-BrdU in Situ DNA Fragmentation Assay Kit (BioVision, USA) was applied for 1 h, followed by incubating the treated plates with 5 µL anti-BrdUFITC antibody. Fifteen minutes of DAPI immunostaining was conducted to identify the nuclei of cardiomyocytes. Images were then taken on an inverted Leica TCS-SP2 AOBS confocal laser-scanning microscope (Leica, Germany). Apoptosis was quantified as the percentage of healthy (no apoptosis) cardiomyocytes, and normalized to the percentage under control condition.

DNA fragmentation was detected using a TUNEL assay and an Apo-BrdU In Situ DNA Fragmentation Assay kit (BioVision, Inc., Milpitas, CA, USA), according to the manufacturer's protocol. A BD FACSCalibur™ was used to perform the analyses. DNA content was quantified using ModFit LT™ software (version 3.0; Verity Software House, Inc., Topsham, ME, USA).

Apoptotic cells were evaluated using the terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), which detects nuclear DNA fragmentation by labeling the terminal end of nucleic acids (Apo-BrdU In situ DNA Fragmentation Assay Kit; BioVision, Mountain View, CA). In brief, the cells were fixed with 1% paraformaldehyde for 15 min on ice and 70% ethanol for at least 30 min at -20 °C. They were incubated with TdT reaction buffer, TdT enzyme, and bromolated dUTP (Br-dUTP) for 60 min at 37 °C and subsequently reacted with the anti-BrdU-fluorescein isothiocyanate (FITC) antibody in the dark for 30 min at room temperature. In addition, the cells were counterstained with propidium iodide (PI) in the dark for 30 min at room temperature. The cells were finally assessed under a fluorescence microscope; the Br-dUTP transferred to the free3'-hydroxyl (3'-OH) groups of cleaved DNA and TdT was detected. Apoptotic cells showed green fluorescence over PI counterstained cells of orange-red fluorescence.