AAT Bioquest

How does the fixed cell viability staining dyes work?

Posted January 26, 2023


Fixed cell viability staining dyes (amine-reactive dyes) work similar to non-fixed dyes, except that instead of binding to DNA, they bind to proteins. They are added to a sample proceeding fixation, the live cells will bind partially to the dye. This is because the fixed dye only binds to the surface of the live cell, which results in extremely dim fluorescence. Dead cells have a much higher affinity to the dye as it can enter the cell and bind to the internal proteins, resulting in very bright fluorescence. In fact, there is a major difference in intensity in fluorescence between live and dead cells, typically greater than 50-fold. Fixed cell viability dyes are used when cells are required to be fixed or permeabilized, for experiments such as intracellular target detection.  

Cells can be fixed with formaldehyde or alcohol when using fixed cell viability dyes. The procedure for using them is simple, there is a 20 minute incubation period and these dyes have a wide array of excitation and emission wavelengths. This makes it simple to find a dye compatible with a specific fluorochrome. Examples of fixed dyes include Horizon proliferation dyes and eFlour fixable dyes.

Additional resources

Evaluation of fluorescence-based viability stains in cells dissociated from scleractinian coral Pocillopora damicornis

Cell Viability Assays

Live or Dead™ Fixable Dead Cell Staining Kit *NIR Fluorescence*