Cellplayer kinetic caspase 3 7 apoptosis assay reagent

Caspase-3/7 activation was assessed by CellPlayer™ Kinetic Caspase-3/7 Apoptosis Assay Reagent (Essen BioScience), which couples the activated caspase-3/7 recognition motif (DEVD) with NucView™488, a DNA intercalating dye. When added to a tissue culture medium, this inert, non-fluorescent substrate crosses the cell membrane and is cleaved by activated caspase-3/7, resulting in the release of the DNA dye and green fluorescent staining of nuclear DNA. Cells were treated with SSP (1 µM), TBT-ITC, and TPT-ITC (500 and 1000 nM) in triplicate in a 96-well plate. Kinetic activation of caspase-3/7 was monitored morphologically using live cell imaging and quantified using the IncuCyte™ FLR object counting algorithm (Essen BioScience).

Live-cell imaging was carried out in a systematic, standardized fashion. Incucyte FLR live-cell fluorescent imaging device (Essen Bioscience) was utilized for continuous monitoring of up to four weeks of 3D culture. Settings were selected to capture one image per well/per hour. These image series are based on phase contrast microscopy combined with green fluorescence, generating image sequences that can be visualized as videos, ideal for investigations of long dose-response and time-course experiments in 3D co-cultures. The cultures were also stained with CellPlayer™ Kinetic Caspase-3/7 Apoptosis Assay Reagent (Essen Bioscience), diluted 1:1000 in culture medium for live-cell imaging of apoptosis. The reagent was mixed in the cell culture medium and pipetted into wells, incubated at +37°C for 72 h, and monitored in real time with the IncuCyte FLR.