IncuCyte Caspase-3/7 and Cytotox Assays

IncuCyte S3 Live-Cell Imaging system (Essen Bioscience, Ann Arbor, MI, USA) was used for kinetic monitoring of cytotoxicity and apoptotic activity of ADV in T cells. Therefore, T cells were seeded in 96-well plates (at a density of 3 × 10⁴ cells/well) and ADV or vehicle control were added in various concentrations. For fluorescence-based cell death analysis, IncuCyte™ Caspase-3/7 Green Reagent (Essen Bioscience, 1.6 µM) and IncuCyte™ Cytotox Red Reagent (Essen Bioscience, 250 nM) were added. Thereafter, plates were placed into the IncuCyte S3 Live-Cell Analysis System at 37°C. Every 3 h, images were taken of each well at 10x magnification for 70 h. Images of green and red fluorescence were monitored and automatically quantified by the IncuCyte S3 v2018B software (Essen Bioscience). Images were analyzed for numbers of green objects (caspase 3/7 positive cells) and red objects (Cytotox positive cells) per well.