Annexin V Assay for Apoptosis in RA

The detection of apoptosis by annexin V assay (BD Pharmingen) was performed on PBMCs isolated from RA patients and HC cultured in serum-free medium for 48 hours as previously described [19 (link)]. Briefly, PBMCs were washed twice with ice cold phosphate buffered saline (PBS) and resuspended in 1X annexin binding buffer containing annexin V as per manufacturer’s instructions (BD Pharmingen). Cells were gently vortexed and incubated at room temperature for 15 minutes in dark. Cell suspension volume was made up to 500μl with 1X annexin V binding buffer and analyzed in flow cytometer (BD FACS Calibur) for annexin V staining.