Cell viability dyes for flow cytometry

Why use cell viability dyes in flow cytometry?

Dead cells can give rise to false positives in flow cytometry experiments due to autofluorescence and increased non-specific binding. It is important to eliminate these dead cells from your flow cytometry data analysis.

​​Several markers, including cellular DNA, cellular esterase, and cellular primary amines, can be used to distinguish between dead and live cells. Using a viability dye can help exclude dead cells and accurately distinguish living cells in your flow cytometry experiments.

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DNA binding dyes

Cell-impermeant DNA binding dyes are used in flow cytometry to effectively exclude live cells from dead ones. The compromised cell membranes found in dead cells facilitate the entry of these dyes into the nucleus. These dyes bind to nucleic acids, allowing for the detection of apoptotic cells and other forms of cell death. Once these dyes are bound to double-stranded DNA, their fluorescence is significantly enhanced which helps to differentiate live cells from dead ones.

Membrane-permeable esterase-activated cell viability dyes

Membrane-permeable esterase-activated dyes are widely used to label live cells. These dyes are non-fluorescent, with a cell-retaining moiety. Upon entering cells, the dye becomes fluorescent following hydrolysis facilitated by intracellular esterases, serving as an indicator of metabolic activity in living cells. These modified dyes are trapped in cells by intact cell membranes. In some cases, hydrolyzed dyes further react with other intracellular components, forming irreversible labels. Dead cells are not stained by these dyes due to the lack of metabolically active esterases and intact cell membranes. These dyes are valuable for monitoring proliferation and metabolic activity in living cells.

Amine-reactive fixable dyes

Fixable viability dyes are commonly used in flow cytometry to stain live cells and distinguish live cell populations from dead ones, even after fixation. Amine-reactive viability dyes react with free primary amines found in cells. As these dyes are membrane-impermeant, live cell intracellular amines exclude the dye, leading to a significant fluorescence intensity difference and clear contrast between live and dead cells. The irreversible reaction of these dyes with amine makes them compatible with post-staining cell fixation and permeabilization. This characteristic makes these dyes useful when following an intracellular staining protocol.

Amine reactive beads are used as compensation controls in flow cytometry methods to correct for fluorescence spillover when using amine reactive dye-based assays.