Apopercentage

Cell apoptosis was assessed by using the fluorimetric kit APOPercentage (Biocolor Ltd., Carrickfergus, UK) following the protocol provided by the manufacturer. This assay has been employed with several adherent cell lines including endothelial cells [37 (link),38 (link)] and uses a dye selectively imported by cells that are undergoing apoptosis. Necrotic cells cannot retain the dye and, therefore, are not stained. HREC cells were treated as previously described in the “cell culture and treatment” section and the apoptosis assay was performed at day 6 of treatment in 96-well black plates (BD Falcon). The APOPercentage dye 3,4,5,6,-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein was added to each well (dilution 1:10) and cells were incubated for 30 more min at 37 °C in a cell incubator. After thoroughly washing, 100 µL of APOPercentage dye release reagent was added to each well, and the cell-bound dye recovered into solution was measured using a GENios plus microplate reader (Tecan) with excitation and emission of 530 and 580 nm, respectively. Results were calculated as the means ± SD of five measurements and expressed as a percentage of untreated control cells.

Apoptosis was measured with the apoptosis assay APOPercentage from Biocolor as previously described [27 (link)]. Briefly, 3 × 10⁴ SKOV3 cells were seeded in gelatine-coated 96 wells and incubated in 100 μl of medium at 37°C, 5% CO2 until confluence was reached (24h). The incubation culture medium was then removed and replaced by 100 μl of fresh medium containing 5% FBS (as control) or 5% ascite in presence or not of H₂O₂ for 6 hours before addition of 5 μl of APOPercentage Dye for 30 minutes at 37°C and 5% CO2. Culture medium was then removed, and cells were gently washed with PBS to remove unbound dye. Then, either red cells were counted under microscopy or 100 μl of Dye releasing reagent was added to the wells to extract bound dye. Wells were shaken for 20 minutes before being read at 540 nm.