The SDS for calcein AM states that it is a non-hazardous substance or mixture. In life science and drug discovery research, calcein AM is commonly used to study enzymatic activity, cell viability, membrane integrity, and long-term cell tracing due to its relatively low cellular toxicity. However, for specific cell lines and experimental conditions, high concentrations of calcein AM have shown to be toxic to cells.

Link: Calcein AM SDS sheet

Hydrolyzed calcein AM (e.g., calcein) is a hydrophilic compound that can be retained in live cells with intact plasma membranes for several hours to days depending upon cell type, cell morphology, or experimental conditions. In a cell-based assay performed by Miles et al., 2016, prostate cancer cells loaded with calcein AM were imaged at 0.5, 2, 6, 12, 24, and 36-hour marks. At the 36 hour mark, cells are still moderately fluorescent; however, there was an increase in background fluorescence. One reason for noise increase is the extrusion of calcein by organic anion transporters (OATs), which play a key role in regulating anion balance. Probenecid, which is an OAT inhibitor, can be added to the calcein AM working solution to improve the intracellular retention of de-esterified calcein and reduce interference.

Fluorescence images of HeLa cells stained with Calcein UltraGreen™ AM (Cat No. 21905) in a Costar black wall/clear bottom 96-well plate. After washing, growth media were added back, and the cells were monitored using a microscope equipped with a FITC filter for up to 24 hours.

Calcein and its derivative calcein AM are generally supplied as a lyophilized solid and must be resuspended in a solution, usually DMSO, before loading into cells. To make a calcein or calcein AM stock solution, reconstitute the dye in high-quality, anhydrous DMSO, such that the concentration is approximately 1 mg/mL. For example, 50 µL of DMSO added to 50 µg of calcein AM will yield a 1 mg/mL stock solution.

For calcein AM, further dilution of the stock solution into an aqueous buffer (e.g., HBSS) is required before loading into cells. As an aqueous solution, calcein AM is susceptible to hydrolysis and should be used within one day.

Positive control: this is a population of healthy, non-treated cells grown in a normal culture environment. Healthy, non-treated cells contain both intracellular esterases to hydrolyze calcein AM into a fluorescent product and an intact plasma membrane to retain the dye. Staining positive controls with calcein AM will result in cells fluorescing green when excited under blue light.

Negative control: this is a population of dead cells, typically fixed with ethanol or formaldehyde. Dead cells do not contain intracellular esterases and, therefore, cannot convert non-fluorescent calcein AM substrates to fluorescent calcein products. Negative controls stained with calcein AM will not produce a fluorescent signal.

It is important to have proper positive and negative controls alongside target samples when performing a calcein AM cell viability assay to track changes and similarities in reported results. All controls should be obtained from the same culture as the target sample.