Cytotoxicity assay measures the ability of a testing compound to cause damage or death to cells in culture.

Cytotoxicity assays are widely used in fundamental research and drug discovery to screen libraries for toxic compounds.

Live/Dead assay is usually used to evaluate cytotoxicity of compounds by determining the number of viable cells remaining after a defined incubation period. It is a convenient and cost-effective method by measuring cell uptake of different fluorescent molecules (e.g., CFSE and 7-AAD) as an indicator of cell viability.
This two-color fluorescence assay allows direct and easy assessment of cell viability in a population over time: it differentially labels live and dead cells with fluorescent dyes (green and red respectively), allowing a rapid quantification of cell viability using flow cytometry or fluorescent microscopy.
Carboxyfluorescein succinimidyl ester (CFSE) is a green fluorescent (λex/ λem = 495/519 nm) and cellpermeable staining dye. It covalently binds to intracellular molecules, thus being retained within cells for extremely long periods. Also, due to this stable linkage, once incorporated within cells, the dye is not transferred to adjacent cells and can be used to monitor cell proliferation, both in vitro and in vivo, due to the progressive halving of CFSE fluorescence within daughter cells following each cell division.
7-Aminoactinomycin D (7-AAD) is a red fluorescent (λex/ λem – DNA = 546/647 nm) intercalating agent that can be used to stain cell nucleic acids. It binds double-stranded DNA, with a high affinity for GCrich regions. 7-AAD is not membrane-permeable, making it useful to differentiate necrotic, apoptotic and healthy cells based on membrane integrity.

The following protocol outlines the steps for a Live/Dead assay to be performed on cellularized inserts cultured in the MIVO® platform.

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