Dyes and Fluorophores 

The Viability/Cytotoxicity Assay Kit for Animal Live & Dead Cells provides green/red fluorescent staining of live and dead cells, respectively. The assay employs two probes that detect intracellular esterase activity in live cells and compromised plasma membrane integrity in dead cells. The esterase substrate calcein AM stains live cells green, while the membrane-impermeable DNA dye Ethidium homodimer III (EthD-III) stains dead cells red. The kit is suitable for detection using fluorescence microscopy, fluorescence microplate reader, or flow cytometry. The assay principles are general and applicable to most eukaryotic cell types, including adherent cells and certain tissues, but not to bacteria or yeast. This fluorescence-based method of assessing cell viability can be used in place of trypan blue exclusion, ⁵¹Cr release, and similar methods for determining cell viability and cytotoxicity. EthD-III (#40050 and #40051) is a proprietary DNA stain developed by Biotium to be a superior alternative to the spectrally similar dye Ethidium homodimer I (EthD-I). EthD-III has higher DNA binding affinity and thus brighter fluorescence (30% brighter than EthD-I).

Spectral Properties
Calcein (end product after hydrolysis of calcein AM):
Ex/Em: 494/517 nm (pH 8)

Ethidium homodimer III (EthDIII):
Ex/Em: 530/620 nm (with DNA)