Erythrosine B (tetraiodofluorescein; ErB) is a negatively charged viability stain commonly used to assess cell viability in a hemocytometer or in adherent mammalian cell cultures. Being considered membrane-impermeable, it preferentially stains dead cells that have lost their membrane integrity (pink color within 1 min of treatment). It is often recommended over Trypan Blue staining for providing a faster and more reliable dead cell staining and for being a generally non-toxic reagent. While exposure to Trypan Blue is toxic to mammalian cells, ErB is often stated to be relatively harmless and suitable for non-terminal cell staining. However, Erythrosine B actually enters live cells and eventually stains them lethally but the speed of this process varies considerably between cell lines and can take from several minutes (U2-OS) to more than 30 minutes (HEK293T).

In the presence of 0.06% w/v (or higher) ErB in serum-free media the adherent U2-OS cells get efficiently stained after 10 min resulting in a pronounced nuclear and a faint cytoplasmic staining while retaining the general cell morphology and adherence. This can be used for a fast terminal staining and counting of adherent U2-OS cells or other cell lines that interact with ErB in the same way.

Lower ErB concentrations in serum-free media often lead to a fast cell disintegration and loss of nuclei in U2-OS cells without staining. The discussed possible destructive and toxic effects of Erythrosine B on mammalian cells should be taken into account when using it as a vital dye. The presence of serum in the media temporarily protects live U2-OS cells from the toxic action of ErB (at concentrations below 0.1% w/v), so it can be recommended when staining dead cells to avoid affecting the live ones.

Prepare the required stock solution and media. Read the Guidelines & Warnings section describing experimental examples and the literature background of the method.