Calcein AM (Calcein acetoxymethyl ester) is a cell permeable, non-fluorescent compound. Upon crossing the cell membrane, calcein-AM is rapidly hydrolyzed by cellular esterases inside live cells. The hydrolysis cleaves the AM group, converting the non-fluorescent calcein AM to a strongly green fluorescing calcein. Because the created calcein is more hydrophilic it is trapped inside the cells with intact membranes. Cells that do not possess active cytoplasmic esterases are unable to convert calcein AM to calcein, and therefore do not fluoresce green. This allows for a quick and easy detection of metabolically-active cells in a sample.

Since calcein-AM does not require DNA binding, it stains all metabolically-active cells and can be used to measure metabolic activity in non-nucleated cells, such as platelets. Because calcein AM is photostable, has low cytotoxicity and does not affect cellular functions, it is a popular stain for the examination of cell vitality and metabolic activity.

The Nexcelom Calcein-AM/PI Cell Vitality and Viability kit enables measurement of the number and concentration of both metabolically active and dead, or non-viable, cells in a population. The software automatically calculates percent vitality/viability based on the number of Calcein-positive (metabolically active) cells, and the number of PI-positive (non-viable) cells.

Assay Principle

Acetoxymethyl ester of calcein (calcein-AM) is a non-fluorescent dye that passively enters cells. In metabolically active cells, the dye is converted by cytosolic esterases into green fluorescent calcein. The fluorescent calcein is retained by cells with intact membranes. Metabolically-active cells fluoresce in the green channel. Propidium iodide (PI) is a DNA-binding dye that enters cells with compromised membranes. Dead (compromised) nucleated cells fluoresce in the red channel.

Calcein AM (Calcein acetoxymethyl ester) is a cell-permeable, non-fluorescent compound. Upon crossing the cell membrane, calcein AM is rapidly hydrolyzed by cellular esterases inside live cells. The hydrolysis cleaves the AM group, converting the non-fluorescent calcein AM to a strongly green fluorescing calcein. The more hydrophilic calcein is trapped inside the cell. Cells that do not possess active cytoplasmic esterases are unable to convert calcein AM to calcein, and therefore do not fluoresce green. This allows for a quick and easy detection of metabolically-active cells in a sample.

When Calcein AM is used in conjunction with PI, it is possible to determine % vitality/viability based on the number of metabolically-active (green fluorescent) and non-viable (red fluorescent) cells in a sample. To enumerate the total number of nucleated cells, the cells were counterstained with Hoechst 33342.