Annexin 5 coupled fluorescein isothiocyanate fitc apoptosis detection kit

The effects of apoptosis, proliferation, and the cell cycle were analyzed after transient overexpression of TMEM62 for 48 and 72 h in OVCAR5 and OVCAR8 cell lines, as described above. The relative percentage of apoptotic cells was assessed by using an Annexin V-Coupled Fluorescein Isothiocyanate (FITC) Apoptosis Detection Kit (BD Pharmingen), according to the manufacturer’s protocol, and DAPI (1 µg/mL) was added for cell cycle analysis. For proliferation analysis, a Click-iT™ Plus EdU Alexa Fluor™ 488 Flow Cytometry Assay Kit was used according to the manufacturer’s protocol.

The effect of the test agents on cell viability was assessed with the MTT assay. Apoptosis was assessed using an Annexin V-coupled fluorescein isothiocyanate (FITC) apoptosis detection kit (BD Pharmingen, San Diego, CA, USA). Briefly, OVCAR-3/ pIRES2-ZsGreen1 -galectin-1 and OVCAR-3/ pIRES2-ZsGreen1 control cells were removed from a six-well plate by incubation with trypsin-EDTA, washed twice in PBS, and re-suspended in 1 ml of Annexin V-binding buffer at 10⁶cells/ml. Annexin V-coupled FITC and propidium iodide were added (each at 5 μl per 10⁵ cells). Samples were mixed gently, incubated for 15 min at room temperature in the dark, and then subjected to flow cytometry to evaluate the number of apoptotic cells. All experiments were repeated at least three times.