Calcein AM (Calcein acetoxymethyl ester), a non-fluorescent, cell permeable compound, converts to the strongly green fluorescent calcein when hydrolyzed by intracellular esterases in live cells (Figure 1).^1-2 It is widely used for determining cell viability since the fluorescence intensity of calcein is proportional to the amount of live cells (Figures 2-4). Calcein is a photostable reagent and is well retained in the cytoplasm of live cells. Calcein is not influenced by intracellular pH. Its bright green fluorescence can be monitored at Ex/Em=494nm /520 nm (Figure 5).

Amongst other reagents, including BCECF, AM and Carboxy-fluorescein diacetate, Calcein AM is the most suitable indicator for staining viable cells due to its low cytotoxicity property. Calcein AM does not affect cell proliferation or viability.^2-3 Calcein AM is used in cell invasion, adhesion, migration and other cell-based assays.

AnaSpec is pleased to provide Calcein AM at two purity grades, >90% or >95%, as determined by HPLC (Figure 6). AnaSpec also offers the SensoLyte^® Calcein Cell Viability Assay Kit - a rapid and sensitive assay to detect viable cells. This kit provides ample reagents (dye and assay buffer) for 1000 assays in a 96-well plate format.

Figure 1. Schematic diagram of viable cell staining

Figure 2. 3T3 cells were seeded into a 96-well plate at the indicated densities. The following day, cells were washed with 1X assay buffer and incubated with Calcein AM for 1 hour. Fluorescence was recorded at Ex/Em=490 nm/ 520 nm (Flexstation 384II, Molecular Devices).

Figure 3. HeLa cells stained with AnaSpec’s Calcein AM fluorescent dye.

Figure 4. MCF-7 cells grown in 8-well slide chamber for 2 days. Cells were washed twice before adding 1 uM Calcein AM (AnaSpec cat# 89202). Images acquired every 3 seconds and total elapsed time on video is 12 min.

Figure 5. Normalized absorption and emission spectra of calcein in PBS buffer (pH 8.5).

Figure 6. Calcein AM Ultra-pure Grade is guaranteed to be at least 95% pure. This HPLC trace shows that for this particular lot, the purity is at >98%.

Related Products

DiIC12(3)

BCECF, AM

CFDA, AM (Carboxyfluorescein diacetate AM)

Calcein

Calcein Blue

Dihydrocalcein, AM

DHL™ Cell Viability and Proliferation Assay Kit *Fluorimetric*

AnaSpec’s Calcein AM, a green fluorescent cell viability indicator, has excellent quality, and is cost-effective.

References

1. Braut-Boucher, F. et al. J. Immunol. Methods 178, 41 (1995).
2. Akeson, AL. et al. J. Immunol. Methods 163, 181 (1993).
3. Weston , SA. et al. J. Immunol. Methods 133, 87 (1990).