How does calcein AM work?

Calcein AM is a non-fluorescent cell-permeable derivate of calcein that is widely used in cell viability measurement. The carboxylic acid groups on calcein are modified with AM (acetomethoxy) groups, which endows calcein AM with high hydrophobicity, facilizing its penetration through cell membrane. Once inside cell, AM groups are hydrolyzed by intracellular esterases. The fluorescent calcein molecule is restored, which is trapped in the cell and emits strong green fluorescence. Since dead cells lack esterase activity, only live cells are labeled and detected. The fluorescence intensity is proportional to esterase activity, thereby this assay is used to evaluate the cell viability.