Fitc labeled annexin 5 and propidium iodide staining solution

Apoptosis of Treg cells was analyzed by seeding Treg cells at a density of 5 × 10⁵cells/well, and starving them overnight in serum free DMEM. The cells were then treated with ZA (0 or 100 μM) for 24 h in DMEM supplemented with 2% FBS. Cells were harvested and incubated with FITC-labeled Annexin V and Propidium Iodide Staining Solution (eBioscience, San Diego, CA, USA). The percentages of apoptotic and necrotic cells were determined by flow cytometry (FACScalibur; BD Biosciences, San Jose, CA, USA). Five parallel samples were measured and 10,000 events were analyzed using Cell Quest Pro software (BD Biosciences, San Jose, CA, USA) or WinMDI software (San Diego, CA, USA). In addition, Treg cells were centrifuged onto microscope slides using a Cytospin2 centrifuge (Shandon Inc., Pittsburgh, PA, USA); the slides were stained with Wright-Giemsa solution and observed by light microscopy (Olympus, Tokyo, Japan).

MDA-MB-231 breast cancer cells were seeded at a density of 5 × 10⁵ cells/well, starved overnight in serum-free medium, treated with ZA (0, 10, 25 and 100 μM) for 48 h at 37 °C in DMEM with 2% FBS, washed with PBS, and incubated with FITC-labeled Annexin V and Propidium Iodide Staining Solution (eBioscience, San Diego, CA, USA). Samples were gently vortexed, incubated for 15 min in the dark, and analyzed within 1 h via flow cytometry using a FACScan flow cytometer (FACScalibur; BD Biosciences, San Jose, CA, USA) to determine percentages of apoptotic and necrotic cells.