The Live/Dead Cell Staining Kit II utilizes two fluorescent dyes, Calcein-AM and Ethidium homodimer III (EthD-III). Calcein-AM penetrates the cell membrane of living cells. Once inside, the cellular esterases present in living cells hydrolyze it to cell membrane-impermeable, green-fluorescent Calcein (emission maximum at 515 nm). In contrast, EthD-III can only pass through the disrupted membrane areas of dead cells where it intercalates with the DNA of the nucleus, emitting red light at 620 nm. Compared with e.g. Propidium Iodide (PI), EthD-III is more stable and emits brighter fluorescent signals. Since both Calcein and EthD-III can be excited at 490 nm, a simultaneous monitoring of viable and dead cells is possible using a fluorescence microscope. At an excitation of 545 nm, the number of dead cells can be monitored.
The Bacteria Live/Dead Staining Kit enables a two-color fluorescent staining on both live bacteria (green) and dead bacteria (red) using the two nucleic acid dyes DMAO and EthD-III. DMAO is a green-fluorescent dye which stains both live and dead bacteria with intact and damaged cell membranes while the red-fluorescing EthD-III only stains dead bacteria with damaged cell membranes. With an appropriate mixture of both dyes, viable bacteria with intact cell membranes are stained green, whereas bacteria with damaged cell membranes fluoresce red. The kit is applicable to most bacteria types for viability and cytotoxicity studies, and suitable for fluorescence microscopy and flow cytometry.
