Fitc propidium iodide apoptosis detection kit

Intracellular ROS was measured by dichlorofluorescein staining assay. Briefly, BMECs were washed with phosphate-buffered saline (PBS) and incubated with fresh DMEM containing 10 μM dichlorofluorescein at 37°C for 35 min; then 1 × 10⁶ cells were harvested and suspended in PBS. The optical density at 450 nm was recorded with a microplate reader (Molecular Devices). In addition, cell apoptosis was detected using an annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection kit (BD Pharmingen, San Jose, CA, USA) according to Wu et al. (13 (link)). Briefly, the cells were resuspended in 1× binding buffer and stained with annexin V–FITC as recommended by the manufacturer. The cells were also stained with PI to detect necrosis. Then the cell suspension was ready for analysis by the flow cytometry (Becton Dickinson, Accuri C6 Plus, CA, USA).

After trypsin digestion and centrifugation, cells were tested by Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) apoptosis detection Kit (all BD Company). Cells were suspended in 400 μL of 1 × AnnexinV-binding solution, and the cell density was adjusted to 1 × 10⁶/mL. Then, 5 μL of Annexin V-FITC staining solution was added and incubated in the dark for 15 min. In addition, 10 μL of PI staining solution was added, and cells were tested using a flow cytometer (BD company, model: FACSCalibur). Cells were divided into 4 quadrants with fluorescein isothiocyanate (FITC) rabbit anti-mouse antibody and PI fluorescence as a two-parameter dot plot: viable cells in the lower left quadrant, early apoptotic cells in the lower right quadrant, late apoptotic cells and dead cells in the upper right quadrant, and mechanically damaged cells in the upper left quadrant. Apoptosis rate = early apoptosis + late apoptosis [27 (link)]