Tunel assay kit

A day after cells were seeded on chambered coverglass, CSG (200 nM) was treated for indicated times under 5% CO₂ at 37 °C. DNA fragmentation was observed with in situ direct DNA fragmentation (TUNEL) assay kit (ab66108; Abcam) according to manufacturer instructions. Briefly, after the media was aspirated, the cells were washed with PBS. For the fixation, 1% paraformaldehyde solution in PBS was added, and incubated for 15 min at 4 °C. After washed with PBS once, 70% ethanol solution in distilled water (DW) was added to the cells, then the cells were incubated for 30 min at 4 °C. As a positive control, the cells were treated with DNase I (Thermo Scientific) for 30 min at room temperature. All cells were washed with the Wash buffer twice. DNA labeling solution was treated for 1 h at 37 °C. The Rinse buffer was added to the labeling solution, and the cells were washed with rinse buffer again. Nuclei was stained with Hoechst 33342 (1:5000 in DW) supplemented with RNase A for 30 min at room temperature in dark. After washed with PBS twice, fluorescent microscopy imaging was performed with DeltaVision imaging system as mentioned above using DAPI/DAPI and FITC/FITC filter sets. Images were analyzed with softWoRx software and ImageJ software.

Formalin-fixed paraffin-embedded tumour sections were stained with haematoxylin and eosin (H&E) to observe morphological changes. PCD in the tumours was detected by TUNEL staining using the TUNEL Assay Kit (ab66110, Abcam), following the manufacturer’s instructions. The sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for observing the nuclei. Quantitative analysis of TUNEL-positive cells was performed on five randomly selected images from each mouse (three mice/group) using Fiji (NIH).

To generate xenograft tumors, 15 million MDA-MB231 breast cancer cells were subcutaneously implanted in the flanks of female SCID mice 8 wk of age, followed by tumor growth monitoring. Mice were orally dosed with either vehicle, 80 mg/kg niraparib daily, IP dosed 100 mg/kg cysmethynil every other day, or the combination of same doses of niraparib and cysmethynil for the treatment, as specified by each study, starting when the tumors reached the stable volume of 100–500 mm³. Tumor growth was followed until termination of experiment, which was when the fastest growing group of tumors, in this case the vehicle-treated control group, reached the mean size of 1,500 mm³. The study protocol was approved by the institutional Animal Care and Use Committee (IACUC). The tumors were isolated after mouse euthanization for imaging and sample preservation, which is by the standard fixation (HT501128; Sigma-Aldrich) and paraffin embedding method. Histology slide preparation and H&E staining were performed by Duke-NUS Histology Service. TUNEL assay to visualize apoptotic cells was per protocol of the manufacture of TUNEL Assay Kit (ab206386; Abcam).

TdT-mediated fluorescein nucleotide (cyanine 3-dUTP) nick-end labeling (TUNEL) was conducted using the TUNEL Assay Kit (ab66110, Abcam, Cambridge, UK) to evaluate the apoptosis of cardiac tissues. the incorporated fluorescein-labeled dUTP was analyzed.

Apoptotic nuclei were identified using a TUNEL Assay Kit (Abcam) following the manufacturer’s protocol. Detection was followed by WT1 staining to label podocytes. Apoptotic cells were counted by a blind observer in 35–45 glomeruli per section, using 3 sections per kidney.