Cellplayer 96 well kinetic caspase 3 7 apoptosis assay

The cells of GS102, GS224 and GS289 were seeded at 5x10³ cells per well in a 96-well plate and incubated with the concentrations of Scriptaid (1.5μM) and LBH589 (15nM) at which synergy was detected. Virus was added at MOI15 or MOI25 followed by the addition of 5μM of reagent of the CellPlayer 96-Well Kinetic Caspase-3/7 Apoptosis Assay (Essen Bioscience). The plates were placed in the IncuCyte imaging system at 37°C in a humidified 95% air/5% CO₂ chamber. Three images/well were taken every two hours in both phase-contrast and fluorescence for 60 hours. Caspase-3/7 activity is presented as counts per well. Experiments were performed in triplicate and means are plotted as percentages of non-treated controls ± standard deviations.

The GS257 GSC cultures were seeded at 5×10³ cells/well in a black 96-wells plate and incubated with the treatment as performed in the viability and Western blot experiments (SAHA, Obatoclax, after 24 hours RTx). Immediately after RTx, the cells were incubated with 5μM of the kinetic apoptosis reagent of the CellPlayer 96-Well Kinetic Caspase-3/7 Apoptosis Assay (Essen Bioscience) and were live imaged by using the IncuCyte system (Essen BioScience) with a 10X objective at 37°C in a humid 95% air/5% CO₂ chamber. The apoptosis-inducer staurosporin (20nM) was used as a positive control. Three fluorescent images/well were collected up to 60 hours post-RTx. The caspase-3/7 activity was presented as counted objects/mm² as measured by the IncuCyte software.