Annexin 5 fitc pi apoptosis assay kit

For apoptosis assays, cells were stained with Annexin V and PI using the Annexin V-FITC/PI apoptosis assay kit (Beyotime, C1062). In brief, hESC cells were digested with 0.5 mM EDTA and centrifuged, and the supernatants discarded. 1 × 10⁵ cells were washed three times with cold PBS and the supernatants discarded. The cells were then resuspended in 195 μl binding buffer from the kit. The cells were then incubated with 5 μl Annexin V-FITC and 10 μl propidium iodide for 20 min in the dark at 25 °C, and then the percentage of apoptotic cells was detected by a flow cytometer (BD FACS Calibur).

The Annexin V-FITC/PI Apoptosis Assay Kit (Beyotime Biotechnology) was utilized to conduct the apoptosis assay. Following the cyclic experimental procedure mentioned earlier, cells were seeded in 6-well plates and exposed to compound XIN-10 for a duration of 48 h. Subsequently, the cells were digested, centrifuged, rinsed twice with PBS, and the supernatants were discarded. Further operations were conducted in a dim setting. The cells were treated with Annexin V-FITC and PI staining solutions in a dark setting for 15 min, and flow cytometry (BD Accuri™C6) was primarily used to detect apoptosis.

The chondrocytes were seeded into 96-well plates and divided into five groups: control, IL-1β, IL-1β+COS, IL-1β+ EVs, and IL-1β+EVs-COS groups. The cells in the IL-1β, IL-1β+COS, IL-1β+EVs, and IL-1β+EVs-COS groups were treated with 10 ng/mL IL-1β for 24 h first, and then with PBS, 320 μg/mL COS, 20 μg/mL EVs, and EVs-COS (20 μg/mL EVs + 320 μg/mL COS), each, for another 24 h, 48 h, and 72 h. Cells in the control group were not treated.
The cell viability of the chondrocytes was examined using a Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology) according to the manufacturer’s recommendations. Briefly, 10 mL of CCK-8 reagent was added to each well and incubated for 2 h. The absorbance at 450 nm was determined using a microplate reader (Multiskan MK3; Thermo Fisher Scientific).
After culturing for 48 h, the cell suspension was harvested and used for the cell apoptosis assay using the Annexin V-FITC/PI apoptosis assay kit (Beyotime Biotechnology). The harvested cell suspension was centrifuged at 1000 rpm for 5 min and resuspended in 1 × binding buffer (100 μL). Afterwards, 5 μL each of Annexin V-FITC and propidium iodide (PI) were added to the cells. After incubation at 22 ± 3 °C in the dark for 15 min, 400 μL of 1 × binding buffer was added to the mixture. Images of apoptosis were acquired using a flow cytometer.

After trypsinization, the cells were collected and centrifuged at 1000 r/min for 10 minutes. Cell apoptosis assay was performed using Annexin‐VFITC/PI Apoptosis Assay Kit (Beyotime) according to the manufacturer's instruction. Finally, we used a flow cytometer (BD FACSCalibur) to detect.

Cell viability of the chondrocytes with different treatments was measured using the Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, Shanghai, China) following the manufacturer’s protocols. The cells were treated with different treatments, and 10 μL of CCK-8 was added. After incubation for 2 h, the absorbance was measured at 450 nm using a microplate reader (Multiskan MK3; Thermo Fisher Scientific).
The Annexin V-FITC/PI apoptosis assay kit (Beyotime Biotechnology) was used to assess cell apoptosis of the chondrocytes with different treatments in accordance with the manufacturer’s recommendations. The cells were harvested and centrifuged at 1000 g for 5 min. After washing with PBS, the cells were resuspended in 1 × binding buffer (100 μL). Next, 5 μL of FITC-Annexin V and 5 μL of PI (50 μg/mL) were added. After incubation at 25°C in the dark for 15 min, 400 μL of 1 × binding buffer was added, and the images were acquired by flow cytometry. The apoptosis rate was calculated using CellQuest software (Becton, Dickinson and Company, NJ, United States).