4.2.2 (c) Detection of Phosphatidyl-serine Exposure by Annexin V-FITC Staining
Phosphatidylserine exposure, an early step of apoptosis induction in protein-stimulated THP-1 macrophages was estimated by Annexin V-FITC apoptosis detection kit (Thermo Scientific). Unstimulated and cells stimulated with Rv2615c and controls- (LPS/Staurosporine) were stained with AnnexinV-FITC and Propidium Iodide (PI) following manufacturer’s protocol and analysed using flow cytometer (BD AccuriC6). Following stimulation time points of 16h, 24h and 48h; macrophages were collected after cold PBS washing and detached by scraping. After washing twice, the cells were re-suspended in binding buffer. An aliquot of 200 µl was mixed with 5 µl of Annexin V-FITC and 5 µl of PI. The mixture was incubated for 10 min at room temperature in the dark. Finally, the cells were analysed by flow cytometry.