Caspase 3 7 luciferase reagent mix

Commercial human PAECs (PromoCell) were cultured in EC media supplemented with 5% FBS, EC growth supplement, and penicillin (50 U/mL)/streptomycin (50 U/mL) and used at passage 3–8. All tissue culture reagents were purchased from ScienCell. For experiments assessing the effect of recombinant HERV-K dUTPase, human PAECs were treated with 10 μg/mL of recombinant HERV-K dUTPase once a day for 3 days, followed by every 3 days up to 20 days. PAECs were evaluated for alterations in cell phenotype, gene, and protein expression at 1, 3, 10, and 20 days after initial treatment using quantitative real-time PCR (qPCR), immunoblotting, and immunofluorescence staining using a confocal microscope (FV1000, Olympus). Apoptosis was assessed on day 1, 10, and 20 following overnight (16 hours) serum withdrawal. Cells were then incubated for 1 hour in 30 μL of Caspase 3/7 Luciferase Reagent Mix (Promega), and total luminescence was measured in a plate reader (BioTek Synergy H1 Hybrid Reader).

For Caspase-Glo^® 3/7 assay cells were seeded in a 96-well plate (7.500 cells/well) in serum-rich medium. Prior to assessing apoptosis, cells were exposed to IL-6 + R with or without Paclitaxel or vehicle, followed by incubation for one hour with 40 μl of Caspase 3/7 Luciferase Reagent Mix (Caspase-Glo^® 3/7 assay; Promega, #G8093, Madison, WI, USA). Total luminescence was measured in a plate reader (Promega GloMax Multi Detection System).