A rapid and simple two-color assay to discriminate between live and dead cells. Compatible with fluorescence microscopy, flow cytometry, and plate reader assays. Live: Ex 495 nm / Em 515 nm | Dead: Ex 528 nm / Em 617 nm ION Vital - Viability kit can be used to quantify live and dead cells within eukaryotic cell suspensions or adherent cultures, 3D cultures, organoids, and some non-fixed tissues. Cannot be used for yeast or bacteria. When following our protocol, the Viability kit provides enough reagents to make 150 mL of working solution, enough for fifteen 96-well plates or 1,500 flow cytometry samples. The actual number of assays will vary according to optimal dye concentrations for your application. Calcein AM is a membrane-permeant, non-fluorescent form of calcein that enters cells passively. Once inside the cytosol of cells, Calcein AM is converted to green fluorescent Calcein by ubiquitous esterases in viable cells, resulting in uniform cytosolic fluorescence (Ex/Em 495 nm/515 nm). Calcein, a polyanionic dye, is membrane-impermeant and is well retained within the cytosol of most healthy cells with intact cell membranes. Ethidium homodimer I (EthD-I) is a membrane-impermeable, high-affinity, nucleic acid stain that is excluded by viable cells with intact cell membranes. When membrane integrity is compromised, a hallmark of dead or dying cells, EthD-I enters the cell and binds DNA, which results in a >30-fold enhancement in bright red, nuclear fluorescence (Ex/Em 528 nm/617 nm).