Fitc conjugated annexin 5 and pi

Cell apoptosis was evaluated by flow cytometry analysis. Cells were seeded in 6-well plates at 1.5×10⁵ cells/well. After 12 h, medium was replaced and cells were transfected with 10 nM siROR1 or siControl and then serum-starved for 12 h. After 96 h of incubation at 37°C in humidified air with 5% CO₂, cells were collected, washed twice with PBS and then resuspended in 100 μl of 1×binding buffer at a concentration of 1×10⁶ cells/ml. Five μl of FITC-conjugated Annexin-V and PI (BD Biosciences, Mountain View, CA, USA) were added to the cells and incubated in the dark for 15 min at RT. 1×binding buffer (400 μl) was added to each tube and apoptosis was measured by flow cytometry (FACSCalibur, BD Biosciences).

Annexin V-FITC/PI double labeling was used to determine the apoptosis of MCF-7 cells cultured in H-DMEM which contained exosomes or PBS in the same volume, as well as the apoptosis-inducing effect of 60 μM 5FU (MEC) on MCF-7 cells cultured in H-DMEM which contained 10% FBS with exosomes or PBS in the same volume. Cells were plated in the 12-well plate and treated the same as above. After 48 h treatment, the cells were harvested by trypsinization and incubated with FITC-conjugated Annexin V and PI according to the manufacturer’s instruction (BD Biosciences). The flow cytometer BD Bioscience Accuri C6 and ModiFit Software were applied for apoptosis analysis. A total of at least 1 × 104 cells were analyzed for each sample. All samples were tested in triplicate, and the data are expressed as the mean ± SD.

Apoptosis extents were determined by flow cytometry using the Annexin V/propidium iodide (PI) double staining. After treating cells with tacrolimus with or without nargenicin A1 or NAC, cells were collected, rinsed with PBS, re-suspended in binding buffer, and stained with fluorescein isothiocyanate (FITC)-conjugated annexin V and PI (BD Pharmingen, San Diego, CA, USA) at room temperature for 20 min in the dark, according to the manufacturer’s instructions. Cell fluorescence intensities were detected using a flow cytometer (Becton Dickinson, San Jose, CA, USA) and data were analyzed using Cell Quest Pro software. A schematic plot was used to display the results: the lower left quadrant represents live cells; the lower right and upper right quadrants represent early and late apoptotic cells, respectively; the upper left quadrant represents necrotic cells. Apoptosis refers to the sum of early and late apoptotic cells.