Fitc anti mouse

Flow cytometry was performed to confirm apoptosis and Fas expression. Cells (1×10⁶ cells/mL) were seeded in 6-well plates and maintained in an incubator (5% CO₂/95% air) at 37℃ for 24 h. The apoptosis assay was conducted using the FITC Annexin V/PI Apoptosis Detection Kit (BD Biosciences, San Diego, CA, USA). The cells were treated with 0, 12.5, 25, or 50 µM DFX, with or without 20 µM FeCl₃, for 24 and 48 hr. The cells were collected and washed twice with cold phosphate-buffered saline (PBS) and resuspended in 1× Binding Buffer (1×10⁶ cells/mL). An aliquot of the cell suspension (100 µL; 1×10⁵ cells) was transferred to a FACS tube, and 5 µL fluorescein isothiocyanate-conjugated (FITC) Annexin V and 5 µL propidium iodide (PI) were added. The cells were vortexed and incubated for 10-15 min at room temperature in the dark. The cells were washed in 1× Binding Buffer and resuspended in 400 µL of 1× Binding Buffer. The cells were harvested 24 hr after DFX treatment to determine CD95 expression. Cells were washed once with PBS and Hanks' balanced salt solution (Gibco/Invitrogen) containing 5% FBS. The cells were stained with CD95 (Fas) (eBioscience, San Diego, CA, USA) for 20 min at 4℃. The cells were then washed and stained with FITC anti-mouse (BD Sciences). All data were analyzed by the FACS Calibur flow cytometer (Becton Dickinson, San Jose, CA, USA).