Annexin 5 fitc pi staining kit

For assessment of apoptosis, an annexin V-FITC/PI staining kit (Dojindo Molecular technologies) was used according to the manufacturer's protocol. Briefly, H9c2 cells were washed with phosphate-buffered saline (PBS), then resuspended in 100 μL of binding buffer at 1 × 10⁶ cells/mL. Aliquots of 10 μL annexin V-FITC stock solution and 5 μL propidium iodide (PI) were added to the cells, and cells were incubated in the dark for 15 min room temperature. Immediately after mixing with 400 μL of binding buffer, the samples were analyzed by flow cytometry with use of a FACSort Flow Cytometer (Becton-Dickinson, Franklin Lake, NJ, USA). Approximately 10,000 cells were counted in each of the samples, and data were analyzed by use of WinMDI software (v2.9, bio-soft Net).

Flow cytometry analysis was used to quantify apoptotic cells after treatment. 1 × 10⁵ cells were seeded in 6‐well plates overnight. After washed with PBS, the cells were incubated with S/sEVs or A/sEVs in FBS‐free media for 3 days. sEVs were dissolved by replacing media and then treated with 10 μm adriamycin for 24 h to induce apoptosis. Both of the attached and floating cells were harvested, and flow cytometry analysis was performed using an Annexin V‐FITC/PI Staining Kit (Dojindo Molecular Technologies, Kumamoto, Japan) according to the manufacturer's instructions. Apoptotic cells were quantified under the detection of a BD FACSCalibur Flow Cytometer (BD Sciences, San Jose, CA, USA).