Cytotoxicity Assays: In Vitro Methods to Measure Dead Cells

Review
In: Assay Guidance Manual [Internet]. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004.
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Excerpt

Membrane integrity is the feature most often used to detect whether eukaryotic cells cultured in vitro are alive or dead. Cells that have lost membrane integrity and allow movement of otherwise non-permeable molecules are classified as non-viable or dead. Detection of dead cells is accomplished by measuring movement of molecules either into or out of cells across membranes that have become leaky and cannot be repaired. A major class of molecules that serve as indicators of dead cells include markers that exist in the cytoplasm of viable cells, but leak into the surrounding culture medium upon loss of membrane integrity. The marker can exist naturally such as an enzyme, or be introduced artificially, such as loading radioactive [51Cr] or a fluorescent marker into viable cells. Artificially introduced markers enable selective detection of target cell cytotoxicity for experiments using more than one population of cells such as cell mediated cytotoxicity. A second class of molecules that serves as an indicator of dead cells is referred to as “vital dyes”. These dyes typically are not permeable to viable cells, but can enter dead cells through damaged membranes. Examples include trypan blue and many fluorogenic DNA binding dyes. Addition of these molecules to cells in culture results in selective staining of the dead cells.

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