Annexin 5 fitc propidium iodide cell apoptosis detection kit

For Th1/CD4⁺ T cells percentage analysis, CD4⁺ T cells were collected and activated with PMA (50 ng/mL) for 2 h, and then monensin (3 μM, a transport inhibitor) was added for an additional 2-h incubation. After harvesting and washing with PBS, CD4⁺ T cells were permeabilized with permeabilization solution (BD Biosciences, USA) for 10 min and fixed with 4% paraformaldehyde for 20 min. For staining, PE-conjugated anti-human IFN-γ antibody (BD Biosciences) was added to cells for 30 min and washed with PBS containing 0.5% FBS. The stained cells were subjected to flow cytometric analysis on a FACSCalibur cytometer (BD Biosciences) and analyzed via CELLQuest software (BD Biosciences).
For apoptosis analysis, ID8 cells were collected and incubated in an annexin V-FITC/propidium iodide (PI) cell apoptosis detection kit (Sigma, USA). Briefly, cells were resuspended with 200 μL binding buffer and incubated with 5 μL annexin V (conjugated with FITC or APC) in the dark for 15 min at 37°C. Finally, the cells were stained with PI or V450 at RT for 15 min, followed by flow cytometric analysis using a FACSCalibur flow cytometer and CELLQuest software (BD Biosciences).

Cell apoptosis was determined using the Annexin V-FITC/ propidium iodide (PI) cell apoptosis detection kit (Sigma–Aldrich, St. Louis, MO, U.S.A.) according to the manufacturer’s instructions. Briefly, cells were seeded into 24-well plates at a concentration of 1 × 10⁶ cells per well and were incubated with FITC-conjugated Annexin V and PI in the dark for 30 min. Finally, apoptotic cells were determined via a flow cytometer (BD Bioscience, San Jose, CA, U.S.A.).