Calcein AM is a non-fluorescent molecule which is freely permeable to the plasma membrane of viable cells.

Store in dark at -20 ℃.

A general protocol is recommended as follow:

1. Prepare a stock solution of Calcein AM at 1mM in DMSO. Keep it in dark.

2. Remove the cell culture media. Wash cells with PBS for 2 times.

3. Add dye diluted Calcein AM in PBS into cells. We recommend staining at a 1-10 μM final concentration of Calcein AM. Since fluorescence intensity may vary based on cell type and experimental conditions, a titration is recommended to determine the optimal dye concentration for each experimental protocol.

4. Incubate cells for 30-60 min at 37°C in dark. Usually 30 min is enough.

5. Remove the supernatant and wash cells with fresh PBS for 2 times.

6. Observe the cells uing a fluorescence microscope with 490 nm excitation and 515 nm emission filters.

Calcein AM is a non-fluorescent molecule which is freely permeable to the plasma membrane of viable cells. Upon cleavage of the lipophilic blocking groups by nonspecific esterases in the cytoplasm, its fluorescent form, calcein, could be released. Calcein AM could be used in study of cell proliferation, cytotoxicity, motility, drug delivery, measurement of intracellular communication, mitochondrial permeability, oxidative activity or intracellular pH, and visualization of cellular structures with fluorescence microscopy, among others.

[1] Miles FL, Lynch JE, Sikes RA. Cell-based assays using calcein acetoxymethyl ester show variation in fluorescence with treatment conditions. J Biol Methods. 2015;2(3):e29. doi: 10.14440/jbm.2015.73. PMID: 26937420; PMCID: PMC4770449.