Annexin 5 fitc pi apoptosis detection kit

Apoptosis was analyzed with the Annexin V-FITC/PI Apoptosis Detection Kit (4A Biotech) following the manufacturer's instructions. Briefly, cells (as specified in each experiment) were washed twice with cold PBS, and then resuspend in the Annexin V Binding Buffer at a concentration of 1-5 × 10⁶ cells/mL. Cell suspension was transferred to a 1.5 mL test tube (100 µL/tube), mixed with 5 µL of FITC Annexin V and incubated at room temperature for 5 min in the dark. The cell suspension was then mixed with 10 µL of Propidium Iodide Solution and 400 µL PBS, and immediately analyzed by FACSCalibur flow cytometer (BD Biosciences).

Annexin V-FITC/PI Apoptosis Detection Kit (4A Biotech, Beijing, China) was used to analyze result of LX2 cell apoptosis. The cells were divided into the following four groups:(1) control, (2) PFD, (3) PPL NPs, and (4) PPL NPs+LIFU. 24 hours later, cells were digested with 0.25% no EDTA trypsin, deposited in centrifuge tube with low-speed, washed with PBS 3 times, then incubated with 5 μl Annexin V-FITC and 10 μl PI into 500 μl binding buffer 15 mins, and kept in dark at 37°C. Analysis of apoptotic cells was done by the flow cytometry procedure (BD Biosciences, Franklin Lakes, NJ, USA).

DLD-1 and LoVo cells were seeded in 6-well plates (2.5×10⁵ per/well) and allowed to adhere overnight. Then, LH alone and LH-robots (20 μM) were added separately, and the cells were incubated for 48 h before being harvested and stained with an Annexin V-FITC/PI Apoptosis Detection kit (catalog no. FXP018; 4A Biotech). FACS DiVa 6.1.3 (BD Biosciences, Franklin Lakes, NJ, United States) was used to analyze apoptosis. Three biological experiments were performed.

GBM cells were plated overnight for adhesion to 6-well plates at a concentration of 2.5 × 10⁵ cells/well. After treating with different doses of NF (0–150 μM) for 24 and 48 h, GBM cells were stained with Annexin V-FITC/PI Apoptosis Detection kit (4A Biotech, Beijing, China). Flow cytometry was used to quantify the apoptotic cells. The experiments were repeated three times.

Apoptosis was detected using the Annexin V-FITC/PI Apoptosis detection Kit (4A Biotech. Ltd, Beijing, China) according to the manufacturer’s instructions. After transfection with shNC or sh2-PBF for 24 hours, ESCA cells were continually cultured in a serum-free medium for 24 h of starvation. Then the cells were collected and washed by cold PBS. Resuspend the cells by 1X binding buffer and adjust the cell density to 1-5 x 106/ml. Take 100 μl of cell suspension in a 5 ml flow tube, add 5 μl of Annexin V/FITC and incubate in the dark for 5 min. Add 10 μl of PI dye solution and 400 μl PBS for flow cytometry analysis. Flowjo software was used for processing results.
For the cell cycle, the transfected ESCA cells were digested with 0.25% trypsin, washed once with PBS and collected at a concentration of 1×106/ml. 1ml of cell suspension was centrifuged and incubated in 70% cold ethanol overnight at 4 °C. After being washed twice with PBS, cells were incubated with a pre-prepared 500 μL PI/RNase: A staining working solution at room temperature for 30-60 min in the dark. Then the cell cycle was analyzed using flow cytometry and Flowjo software.