Annexin 5 fluorescein isothiocyanate fitc propidium iodide apoptosis detection kit

EECs were planted in 6-well plate (5 × 10⁵ cells/well) and cocultured with or without hucMSC-Ex for 24 h. The annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Beyotime Biotechnology, C1062, China) was used to determine apoptosis according to the manufacturer's guidelines. Cells were sorted within 1 h by a FACScan flow cytometer (BD FACS Calibur).

Apoptotic cells were analysed using an annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (Beyotime, Shanghai, China). The primary hepatocytes were first isolated from the mouse liver using a previously described procedure^{48 (link)}. Then, cells were seeded in plates and cultured overnight in DMEM supplemented with 10% FBS. Hepatocytes were transfected with Ad-HIPK2 or Ad-vector (2 × 10³ plaque-forming units per well) for 16 h, followed by an incubation with 0.5 μg/mL LPS for another 12 h. Cells were harvested and centrifuged at 250 g for 5 min at 4 °C and resuspended in cold binding buffer (10 mM HEPES buffer, pH 7.4, 5 mM KCl, 150 mM NaCl, 1.8 mM CaCl₂, and 1 mM MgCl₂). Next, hepatocytes were stained with FITC-labelled 25 ng/mL FITC-labelled annexin V in the dark for 40 min, followed by an incubation with 50 ng/mL propidium iodide for 5 min. Samples were analysed with a Becton Dickinson flow cytometer (USA). The results were analysed with flow cytometry software. In the present study, apoptotic cells were stained with annexin V but not PI. Hepatocytes stained with both annexin V and PI were considered necrotic or late apoptotic cells.

Cell cycle and cell apoptosis were evaluated using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) Apoptosis Detection Kit (Beyotime). For cell cycle analysis, transfected PCOS GCs and KGN cells were collected and fixed in ice-cold 75% ethanol overnight at 4 °C. Next, cells were washed, resuspended (2 × 10⁵ cells/mL) and incubated with PI (Beyotime) and RNase (Solarbio) for 30 min at room temperature. The cells were then analyzed using a flow cytometer (BD Biosciences, San Jose, CA, USA). For cell apoptosis analysis, transfected PCOS GCs and KGN cells were harvested, washed, resuspended and the incubated with 5 μL AnnexinV-FITC (Beyotime) and 10 μL PI (Beyotime) for 15 min to stain cells in the dark. Finally, cell apoptosis was assessed with a flow cytometry (BD Biosciences).