Tunel apoptosis detection kit

The apoptosis rate of NP cells was detected using a fluorescein isothiocyanate–annexin V and propidium iodide (FITC/PI) Apoptosis Detection Kit (Yeasen, China) according to the manufacturer's instructions. NP cells were pre-treated with 100 ​μg/mL of PLTs and 100 ​μg/mL of PEVs for 24 ​h, respectively. Then, 400 ​μM ​H₂O₂ were added for 4 ​h to induce apoptosis in NP cells. NP cells were centrifuged and then resuspended in 300 ​mL of PBS supplemented with annexin V–Alexa FITC (5 ​μL) and PI (10 ​μL). Following incubation (10–15 ​min, 20 ​°C) in the dark, cell apoptosis was detected using a flow cytometer after adding 400 ​μL 1 ​× ​binding buffer. The total apoptosis rate is the sum of the early stage of apoptosis (Annexin V–positive/PI-negative) and late stage of apoptosis (Annexin V–positive and PI-positive). For apoptosis phenotype, NP cells were labeled with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) Apoptosis Detection Kit (Yeasen, China). The changes in apoptosis levels in the NP cells were detected by fluorescence microscopy, and the TUNEL-positive cells were counted. We analyzed the results using FlowJo software. All experiments were performed with four replicates each.

To stain TdT‐mediated dUTP Nick‐End Labeling (TUNEL) cells, we used the TUNEL Apoptosis Detection kit (YEASEN, CN) following the manufacturer's protocol. Counterstaining with 4′,6‐diamidino‐2‐phenylindole (DAPI) (YEASEN, CN) was performed to visualise nuclei. Sections were photographed under a fluorescence microscope (Olympus, IX70, JPN).

A TdT‐mediated dUTP nick‐end labeling (TUNEL) Apoptosis Detection Kit (Alexa Fluor 488) (Yeasen, China) was used to detect apoptotic cells in the heart tissue and NRCMs. All procedures followed the manufacturer's instructions.

Further analysis of apoptotic cells was conducted by TUNEL Apoptosis Detection Kit (Yeasen, Shanghai, China). Human SSCs were treated with miRNA mimics control, miR-663a mimics, miRNA inhibitor control, miR-663a inhibitor, miR-663a inhibitor and NFIX siRNA 3, control siRNA, or NFIX siRNA 3, according to the methods mentioned above. The cells were fixed with 4% PFA for 25 min at 4°C. After several washes, the cells were incubated with Proteinase K (20 μg/mL) and 1× DNaseI Buffer for 5 min at room temperature. Cells were then treated with 10 U/mL DNaseI for 10 min at room temperature and followed by washes in deionized water. These cells were incubated with 1× Equilibration Buffer for 30 min at room temperature and labeled by Alexa Fluor 647 in buffer premixed with TdT Enzyme for 60 min at 37°C. After being washed with PBS, the cells were finally stained with DAPI and analyzed under a fluorescence microscope (Nikon, Tokyo, Japan).

TUNEL staining in HaCaT cells and mouse
skin tissue was performed to assess apoptosis using a TUNEL apoptosis
detection kit (Yeasen, China) following the manufacturer’s
instructions. The nuclei of all cells and apoptotic cells appeared
in blue and green, respectively.

A TUNEL Apoptosis Detection kit (FITC; Yeasen) was used to detect PC12 cell apoptosis. At the same time, the nuclei were counterstained with a DAPI staining solution (1 µg/ml; Beyotime) at room temperature for 3-5 min. A laser-scanning confocal microscope (Nikon) was used to examine the cells.