Annexin 5 propidium iodide staining

Apoptosis was analyzed by Annexin V/Propidium iodide (PI) staining (BD Pharmingen) according to manufacturer's instructions. In brief, MDA-MB-231 cells were treated with DKG (10 mM, 24-hour), trypsinized, washed with PBS and re-suspended in binding buffer. Approximately 1×10⁵ cells were stained with Annexin V-FITC and PI, incubated at room temperature for 15-min, and analyzed by Accuri C6 Flow Cytometer (BD Biosciences).

To investigate the effects of NBP on hBMSCs cell apoptosis, cells were harvested in 6-well plates at a seeding density of 2 × 10⁵ cells per well. The assessment of hBMSCs apoptosis was performed through Annexin V/propidium iodide (PI) staining (BD Biosciences Pharmingen, CA, USA), following the provided instructions from the manufacturer. The apoptosis rate in hBMSCs was assessed at various time points (0, 4, 7, and 10 min) following 24h of NBP treatment. Briefly, cells were collected by 0.5% EDTA-free trypsin (Hyclone SH30042.01) and washed twice with cold PBS. The cells were then re-suspended in 1 × binding buffer. Afterward, the cells were stained with 5 µl Annexin V-FITC and 5 µl propidium iodide for 15 min in the dark at room temperature, and flow cytometry (BD Biosciences, USA) was performed within 1h.

Apoptosis was detected with Annexin V/propidium iodide (PI) staining (BD BioSciences) according to the manufacturer’s instructions. In total, 10, 000 cells were counted by flow cytometry using a fluorescence-activated cell sorter (FACS, Becton-Dickinson, San Jose, CA, USA). The resulting data were analyzed using Summit 5.2 software (Beckman Coulter Inc., Miami, FL, USA).