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Modifications Required for ATP and Caspase Detection Assays Applied to 3D Cell Spheroids Poster

Part # PS254

Abstract

Terry Riss, Michael P. Valley, Kevin R. Kupcho, Chad A. Zimprich, Donna Leippe, Andrew Niles, Jolanta Vidugiriene, Brad Hook, James J. Cali, & Dan F. Lazar
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI, 53711

We investigated whether cell viability and apoptosis assays designed for 2D monolayers of cells would work effectively with 3D spheroid models. Cell viability assay reagents that measure ATP contain detergent to lyse cells and release ATP. Some commercial reagents were found to have a low recovery of ATP from spheroids compared to acid extraction accepted as the gold standard. Reformulation of ATP assay reagent to contain higher amounts of detergent (CellTiter-Glo® 3D Assay), increasing mixing time using a plate shaker, and using a longer incubation time in the presence of lytic reagents resulted in improvements in extraction of ATP from large 3D cell spheroids. Although caspase-3 assay reagents (Caspase-Glo® 3/7 Assay) contain detergent to lyse cells, large increases in detergent concentration to achieve lysis of large 3D spheroids was not possible because of damage to the caspase-3 enzymatic activity. Modification of the caspase-3 assay protocol to include 5 minutes of plate shaking followed by 25 minutes incubation resulted in improved recovery of active caspase from 3D spheroids.

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