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Multiplexing Cell-Based Assays Using 3D Culture Models
Poster Title: Multiplexing Cell-Based Assays Using 3D Culture Models
Submitted on 25 May 2017
Author(s): Terry Riss, Sarah Duellman, Mike Valley, Kevin Kupcho, Brad Hook and Andrew Niles
Affiliations: Promega Corporation, 2800 Woods Hollow Rd, Madison, WI 53711
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Poster Information
Abstract: The physical nature and size of 3D cell culture models can be much different than cells grown as a monolayer on a plastic surface. Applying off the shelf commercial assay reagents that were originally designed for use with monolayers of cells can lead to artifacts if incomplete cell lysis or incomplete reagent penetration occurs in the larger 3D structures. Those problems may be further amplified when attempting to combine (multiplex) more than one assay chemistry to interrogate the same sample comprised of 3D cell structures. We have designed modified cell health assay formulations and protocols to overcome some of the problems encountered with assaying 3D culture models. We have also tested combining/multiplexing different assay combinations on the same samples of individual spheroids formed using the hanging drop method. We have combined a novel real time cell viability assay with: measuring cell death using a DNA binding dye, measuring firefly luciferase reporter activity to detect cell stress events, and extraction of RNA to perform gene expression analysis. The parameters necessary to validate each multiplex assay combination and the advantages and disadvantages of each method will be described. Summary: Multiplexing cell-based assays is possible using 3D culture models that are larger and more complex than monolayers

Real-time detection methods to measure live or dead cells provide much flexibility for multiplexing

All multiplexed assay combinations should be verified using appropriate controls for each 3D cell culture model.
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