One step tunel apoptosis assay kit

For this experiment, 5 × 10⁵ cells were grown on coverslips. Cell apoptosis was determined using the one-step TUNEL apoptosis assay kit (Roche, 12156792910) according to the manufacturer’s instructions. Following TUNEL staining, the slides were stained with DAPI (Solarbio Beijing, C0060) to highlight the nuclei with fluorescence. Slides were observed under an LSM 510 META Olympus Confocal Laser Scanning Microscope using 488-nm excitation and 530-nm emission, and the images were captured and quantified by image analysis software, ImageJ.

The One-Step TUNEL Apoptosis Assay Kit (Roche, Mannheim, Germany) was used to evaluate DNA fragmentation in vivo. Images were taken using a Nikon ECLIPSE Ti microscope (Nikon, Melville, NY, United States). The apoptosis ratio of HBMECs was determined using a PI/Annexin V-FITC kit (Invitrogen). Next, a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, United States) was employed for analysis according to the kit’s manual.

The TdT‐mediated biotin‐16‐dUTP nick‐end labelling (TUNEL) assay was performed using a One‐Step TUNEL Apoptosis Assay kit (Roche) to detect apoptotic cells in the mouse brain. In brief, 4‐μm‐thick paraffin sections were deparaffinized, hydrated, treated with proteinase K for 20 minutes and subsequently incubated with a mixture of a fluorescently labelled solution of dUTP and the TdT enzyme at 37°C for 1 hour in a humidified atmosphere. As a positive control, sections were incubated with DNase I for 10 minutes at room temperature (25°C) before the fluorescent labelling procedure. Negative controls were incubated with dUTP for 10 minutes at room temperature (25°C). Subsequently, the samples were treated with diaminobenzidine, counterstained with haematoxylin (to identify the cell nuclei). The cells were then dehydrated in a gradient ethanol series, vitrified with dimethylbenzene and mounted with neutral balsam.

DNA fragmentation in vivo was detected using a one-step TUNEL Apoptosis Assay KIT (Roche, Mannheim, Germany). The images were captured with a Nikon ECLIPSE Ti microscope (Nikon, Melville, NY, USA). The apoptotic rates of the H9C2 cells treated with TBHP and bFGF were measured using a PI/Annexin V-FITC kit (Invitrogen) and then analysed by a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA) according to the kit's manual.

In situ DNA strand breaks were detected using the One Step TUNEL Apoptosis Assay Kit (Roche, Mannheim, BW, Germany) according to the manufacturer’s protocols described previously [6 (link)]. Briefly, 48 h after siNC or siUSP22-1 transfection, GC cells were seeded onto glass slides and fixed with 80% glycerol at room temperature for 60 min. Then, the cells were rinsed with PBS (pH 7.4), and permeabilized with 0.1% Triton X-100 in PBS. The permeabilized cells were then incubated with FITC-labeled terminal deoxynucleotidyl transferase (TdT) nucleotide mix at 37°C for 60 min in the dark. Then, the cells were rinsed twice with PBS and counterstained with 10 mg/ml DAPI. In tissues, the specimen were fixed with 4% formalin overnight, Then, the fixed tissues were embedded in paraffin, cut into 4 μm thick sections and mounted onto polylysine-covered slides. After incubation in xylene to remove paraffin, the tissue sections were rehydrated by incubation in a graded series of ethanol solutions. Then, the tissue sections were rinsed with PBS, incubated with FITC-labeled TdT nucleotide mix at 37°C for 60 min in the dark, and counterstained with 10 mg/ml DAPI. FITC-labeled TdT was omitted in the nucleotide mix of the negative control. The percentage of TUNEL-positive cells was estimated in all samples (cells and tissues) using fluorescence microscopy (Carl Zeiss).