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Multiplexing Cell-Based Assays Using 3D Culture Models

Multiplexing Cell-Based Assays Using 3D Culture Models content piece image
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.