Annexin 5 fluorescein isothiocyanate fitc and propidium iodide staining

In order to measure cell death, the RT4 cells were seeded in 6-well culture plates at a density of 2×10⁵ cells per well. The RT4 cells were transiently transfected with wild-type MPZ or mutant MPZ for 24 h, then treated with the ASA compounds (1-100 μM) for 24 h and harvested. Annexin V fluorescein isothiocyanate (FITC) and propidium iodide (PI) staining (BD Biosciences Pharmingen) was performed by incubating the cells in the dark for 15 min at room temperature in binding buffer (10 mM HEPES, 140 mM NaCl, and 2.5 mM CaCl₂, at pH 7.4) saturated with Annexin V FITC and PI. Following incubation, the cells were washed with cold PBS, pelleted and analyzed by a FACS VERSE analyzer (BD Biosciences). We determine the number of live, apoptotic and necrotic cells by counting the numbers of Annexin V^-/PI^- cells, Annexin V⁺/PI^- cells, and Annexin V⁺/PI⁺ cells, respectively.