Annexin 5 apc

1ˆ10 (Luo et al., 2011 (link)) cells were harvested and washed with PBS. Cells were centrifuged and resuspended in binding buffer contains Annexin-V APC (KeyGEN BioTECH, China) and propidium iodide(PI) (KeyGEN BioTECH, China), which were excited at 633 and 488 nm and emitted fluorescence at 660 and 610 nm, respectively. Flow cytometric assays were performed and over 10,000 cells per sample group were collected. The percent of Annexin-V positive population in glioblastoma cells was calculated as an indication of apoptosis.

For the apoptosis assay, 3×10⁵ cells in 500 µl of binding buffer were incubated with 5 µl of Annexin V-APC and 5 µl of propidium iodide (KeyGEN Biotech, Nanjing, China) for 10 min. Then, flow cytometry was used to measure cell apoptosis. For the cell cycle assay, the cells were immobilized with ethanol and centrifuged at 800 rpm for 5 min, and the supernatant was discarded. The cells were cleaned twice with PBS. Then, 150 µl of PI (propidium iodide) (KeyGEN Biotech, Nanjing, China) working solution was added to these cells for 30 min at 4 °C. Next, flow cytometry was used to measure the cell cycle.

Cells were removed from tissue culture plates with trypsin and washed twice in ice-cold PBS. A total of 5 × 10⁵ cells were resuspended in 500 µL of binding buffer (KeyGen, China). This was followed by the addition of 5 µL of Annexin V-APC (KeyGen) and 5 µL of the counterstain, 7-amino actinomycin D (7-AAD). The mixture was incubated at room temperature for 10 min in the dark, followed by quantitative analysis of apoptosis using a FlowSight flow cytometer (Merck, Germany).

Transfected and tBHP-exposed NPCs were incubated with a mixture (100 μg/mL) of 5 μL Annexin V-APC and 1 μL PI for 15 min according to the kit instructions (Keygen Biotech, China). Apoptotic cells were examined using the FACScan flow system (Becton Dickinson, San Diego, CA) and analyzed employing FlowJo software.