3.7. Real-Time-GloTM Annexin V Apoptosis and Necrosis Assay (Promega, Catalog no. JA1011)

The assay was performed according to the manufacturer’s instructions (Promega, Walldorf, Germany). Cells were seeded on a 96-well plate (5000 cells per well for SKOV3 cell lines, 10,000 cells per well for A2780 cell lines). Complex 7 was dissolved in DMSO and diluted in RPMI 1640 medium to reach the stated concentrations, which were added in 100 μL. Untreated cells in RPMI 1640 medium served as control. Next, 100 μL of the 2X detection reagent (containing 12 mL RPMI 1640 medium, 24 μL Annexin NanoBiT^® substrate, 24 μL CaCl₂, 24 μL necrosis detection reagent, 24 μL Annexin V-SmBiT and 24 μL Annexin V-LgBiT) were added per well. The plate was shaken for 30 s, and both luminescence and fluorescence measurements were conducted at various time points over 70 h with a Tecan plate reader M200pro. The excitation wavelength was set at 485 nm, the emission wavelength was set to 530 nm.