Calcein AM/PI Double Staining Kit

Calcein AM is the addition of an acetyl methoxy methyl ester (AM) group to Calcein, which increases its hydrophobicity, allowing it to easily penetrate live cell membranes. Calcein AM itself is non-fluorescent. Once inside the cell, it is hydrolyzed by intracellular esterases to produce Calcein, a highly negatively charged, polar molecule that cannot pass through the cell membrane and thus stays inside the cell. Calcein emits strong green fluorescence (Ex/Em = 494 nm/517 nm). Since dead cells lack sufficient esterases, they cannot or only weakly produce Calcein, resulting in strong green fluorescence in live cells and minimal or no staining in dead cells. Dead cells, having compromised membrane permeability, allow Propidium Iodide (PI) to enter and bind specifically to double-stranded DNA, producing intense red fluorescence (Ex/Em = 535 nm/617 nm) to mark dead cells. Thus, the combined use of Calcein AM and PI enables simultaneous dual fluorescence staining of live and dead cells, useful for assessing cell viability and cytotoxicity.