Bms500fi 300

The cells of passages 3 or 5 were harvested and suspended in 200 μL Binding Buffer (Invitrogen, BMS500FI-300) to a final density of 3×10⁵ cells/mL. Cell suspension (195 μL) was incubated with 5 μL Annexin V-FITC (Invitrogen, BMS500FI-300) for 10 min at room temperature. After a centrifugation, the cells were washed with 200 μL Binding Buffer and resuspended in 190 μL Binding buffer, then incubated with 10 μL Propidium Iodide (20 μg/mL, Invitrogen, BMS500FI-300). Cells were then analyzed using a flow cytometer (BD FACSCalibur).

Cells were washed three times in PBS and stained with an apoptosis detection kit (BMS500FI/300, eBioscience) consisting of FITC-conjugated annexin V and propidium iodide (PI) following the vendor’s recommendation. The stained samples were assayed using an LSR II (BD) flow cytometer and analyzed using FlowJo software.

Apoptosis of cells transfected with plasmids encoding flag-ORF7b or treated with inhibitors was detected using the Annexin V-fluorescein isothiocyanate (FITC) Apoptosis Staining/Detection kit (BMS500FI-300; eBioscience, San Diego, CA, United States). Cells (1 × 10⁵) from each well were collected 18 h after transfection, and 500 μl of binding buffer was added to resuspend them after washing thrice with phosphate-buffered saline. Then, cells were mixed with 5 μl of Annexin V-FITC and 2 μl of PI (Propidium Iodide, PI). Then, the cells incubated in the dark for 10 min at room temperature. Cell apoptosis was detected by flow cytometry using a CytoFLEX^™ system (Beckman Coulter, Fullerton, CA, United States) and the experiment was repeated independently thrice.