Annexin V-FITC Apoptosis Detection Assay

The cell death type induced by compounds was determined with an Annexin V-FITC Apoptosis detection kit (Immunostep, Salamanca, Spain) following the manufacturer’s instructions [56 (link)]. Cells were seeded in 12-well plates at 1 × 10⁶ cells per well and treated with compounds at IC₅₀ concentrations for 6 and 24 h. Then, cells were washed, resuspended in PBS (Phophate-Buffered Saline), and 5 μL of Annexin V-FITC and Propidium Iodide (PI) were added to each tube. Cells were incubated for 15 min in the dark and analyzed by flow cytometry using the ImageStreamMKII instrument (Amnis Corporation, LuminexCorp, Austin, TX, USA). The fluorescence of 10,000 events was analyzed with IDEAS Application 6.0 software (Amnis Corporation, LuminexCorp). The percentages of apoptotic cells, including early apoptotic cells (Annexin-FITC positive and PI negative) and late apoptotic cells (Annexin-FITC and PI-positive), and necrotic cells (Annexin-FITC-negative and PI-positive), were calculated. To further confirm if apoptotic cell death was occurring, SH-SY5Y cells were preincubated with the pan-caspase inhibitor Z-VAD-FMK (Merck) for 24 h. Then, the assay was carried out as described above. Staurosporine (STS) (Merck) was used as a positive control in all the experiments [56 (link)].