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Design and Validation of Bioluminescent Assays for 3D Cell Culture Models

Part # PS202


Terry L. Riss, Michael P. Valley, Chad A. Zimprich, Andrew L. Niles, Kevin R. Kupcho and Dan F. Lazar
Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711
Abstract #226

Cells cultured in 3D model systems often acquire relatively large in vivo-like structures compared to the thickness of a 2D monolayer of cells grown on standard plastic plates. Multicellular 3D culture systems containing more than one cell type and exhibiting formation of a complex extracellular matrix represent a more physiologically relevant environment, yet provide a challenge for assay chemistries originally designed for measuring events from monolayers of cells. There is an unmet need for guidelines for design and verification of convenient and effective assays useful for larger 3D microtissues. Critical factors to consider for each model system include effective penetration of detection reagents and/or complete lysis of microtissue structures using combinations of detergent and physical disruption. We have developed an improved reagent formulation for bioluminescent detection of ATP for measuring cell viability. The improved formulation demonstrates more effective lysis of large microtissues. Results from modifying assay procedures to include more rigorous physical disruption of microtissues will be presented for measuring caspase activity for detecting apoptosis and cell stress assays to detect mechanisms leading to cytotoxicity.

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