Annexin 5 fitc pi apoptosis detection kit

SKOV3, COC1/DDP cells were pre-treated with 10 µg/mL of eNK-EXO, DDP or eNK-EXO-DDP for 24 h. Cell apoptosis was evaluated by flow cytometry analysis using Annexin V-FITC/PI apoptosis detection kit (Absin, China). Briefly, cells were washed in PBS twice and suspended in 1× binding buffer, incubated in the dark with FITC-Annexin-V for 10 min, followed by PI for 5 min before flow cytometry analysis. Cell cycle assay was carried out using cell cycle detection kit (KeyGEN Biotech, China). Briefly, cells were fixed by 70% ethanol overnight, washed in 1×PBS and treated with RNase-containing propidium iodide for 30 min before flow cytometry analysis. To measure cell proliferation, SKOV3 cells were labeled with 2.5 μM CFSE dye solution for 20 min in the dark and then cultured with the same conditions as above for 48 h after removing free dye. Stained cells were collected on a FC500 flow cytometer (Beckman Coulter, USA) and the data was analyzed by the FlowJo-10 software.

The apoptosis was performed using the Annexin V-FITC/PI apoptosis detection kit (Absin Bioscience Inc., Shanghai, China). Briefly, cells (3 × 10⁵) were incubated with 12e (0–100 nM) for 24 h, and then harvested by centrifugation, washed with ice-cold PBS twice, and resuspended in binding buffer. Staining was started by adding Annexin V-FITC (5 μL) and PI (5 μL) followed by incubation for 10 min at room temperature in the dark. Then, samples were immediately analyzed by NovoCyte flow cytometer (ACEA Biosciences, San Diego, CA, USA).

Apoptotic cell death in kidney sections was determined using TUNEL staining (Promega, Madison, WI, USA). Briefly, kidney sections were deparaffinized and pretreated with 0.1 M sodium citrate, pH 6.0, at 65 °C for 30 min and then incubated with a TUNEL reaction mixture for 1 h at 37 °C in a dark chamber; the nuclei were labeled by DAPI. Positive staining with nuclear DNA fragmentation was detected by fluorescence microscopy (Zeiss, Germany). Each section was selected for ten representative fields randomly and the TUNEL-positive cells per mm² were counted.
Flow cytometric analysis was performed using the Annexin V-FITC/PI apoptosis detection kit (abs50001, Absin, Shanghai, China) to evaluate the percentage of apoptotic cells. Cells were harvested, washed twice with cold PBS, and resuspended in 100 μL of binding buffer. Cells were stained with 5 μL of annexin V-FITC for 15 min and 5 μL of PI for 5 min at room temperature in the dark, and then measured by laser eight-color flow cytometer (FACSCalibur, BD Biosciences, San Jose, CA, USA) and quantified using FlowJo 7.6 software.

The Annexin V-FITC/PI apoptosis detection kit (Absin Bioscience, Shanghai, China) was employed according to the manufacturer’s protocol. LO2 and SMMC-7721 cells were plated in 6-well plates at a 1.5 × 10⁶ cell/well concentration. When cells reached the logarithmic stage, they were treated with 2 mL liposomes or 10 mL HCPT injection (using the same medication as for the MTT experiment) at a concentration of 1 μg/mL. The cells were trypsinized (without EDTA) and rinsed three times with PBS after 48 hours. The cells were then resuspended in 300 μL of 1 × binding buffer and stained with 5 μL of Annexin V-FITC and 5 μL of 100 μg/mL PI (dark operation). After 15 minutes of incubation, each resuspension solution received 200 μL of fresh 1 × binding buffer, and the cells were examined using flow cytometry and the software FlowJo.