AAT Bioquest

Calcein-Based Cell Viability Assays

Cell viability assays are an essential part of biological research. For example, cell viability assays are used in cancer studies to determine the cytotoxicity of a particular drug on a subset of cells, and by extension, provide a measure of the proliferation of cancerous cells. Cell viability assays are also critical in the study of cytomembranes, whether it be a mitochondrial membrane, a vesicular membrane, or the membrane of the cell itself. Many researchers are generally interested in quantifying the number of viable cells after an experimental procedure, to gauge the effect (or lack thereof) of such procedures on cell viability. Given the importance of such assays, therefore, many different methods have been developed to quantify cell viability. One particularly prevalent compound is calcein.

Calcein is a fluorescein derivative which was originally implemented as a calcium indicator. It has since been developed for use in cell viability assays. To deliver the fluorescent calcein probes into cells, a hydrophobic group, such as acetomethoxy (AM), is attached. With this reaction, calcein becomes calcein AM- a hydrophobic, nonfluorescent compound. This form of calcein allows it to readily pass through lipid membranes, whether it'd be a vesicular membrane or the cell membrane itself. Then to reach its active state, calcein AM is hydrolyzed by esterases which remove the acetomethoxy, returning the probe back to its calcein form. In this new state, the probe becomes hydrophilic, which allows it to be retained in the cell. Furthermore, it gains a strong fluorescence, which maximally excites at 495 nm and emits at 515 nm. Since calcein AM is a fluorometric assay, it has both a greater sensitivity and a greater range of sensitivity than colorimetric assays, such as MTT.

Fluorescence images of HeLa cells

Fluorescence images of HeLa cells stained with Calcein UltraBlue™ AM (Upper row, Cat# 21908) or Calcein Blue AM (Lower row, Cat# 22007) in a Costar black wall/clear bottom 96-well plate. Left: Live HeLa cells in HH buffer; Middle: Live HeLa cells in medium; Right: Fixed HeLa cells.

Image of CPA cells
Image of CPA cells in a 96-well Costar black wall/clear bottom plate stained with CytoCalcein™ Violet 500 (Cat# 22013, Ex/Em = 405/500 nm, 405 Violet filter).
Aside from its good solubility and greater sensitivity, calcein AM also benefits from having low cytotoxicity during its use. This derives from the fact that very little calcein is needed to achieve bright imaging results. Since less calcein AM is needed, the impact on cellular functions, such as proliferation, and organelle structures, such as mitochondria, is minimized. Additionally, the turnaround time is very short for calcein AM assays, meaning that cytotoxicity of calcein is further reduced. Unlike some assays, which have a procedure time of four to six hours, calcein based assays can be completed in less than two hours. Lastly, one important benefit calcein has is that it can be used in multicolor analysis. Dyes such as CytoCalcein™ Violet 450, CytoCalcein™ Violet 500, CytoCalcein™ Blue 550 and CytoCalcein™ Blue 600 have been developed for flow cytometric applications. CytoCalcein™ dyes exhibit similar biological properties to calcein AM. They are optimized for the excitation wavelengths of a variety of flow cytometers, providing additional colors for flow cytometric analysis of live cells. CytoCalcein™ Violet 450 and CytoCalcein™ Violet 500 are well excited by violet lasers (405 nm) and emit fluorescence at 450 nm and 500 nm respectively. CytoCalcein™ Blue 550 and CytoCalcein™ Blue 550 are well excited by blue lasers (488 nm) and emit fluorescence at 550 nm and 600 nm respectively. The wealth of colors allows for multiple variables to be probed at once. For example, calcein can be used in conjunction with GFP to probe a cell in two dimensions. Such multi-color analysis cannot be achieved with colorimetric assays such as MTT and is a unique property of calcein based dyes.

Table 1. Calcein-Based Cell Viability Probes

Cat No.
Product Name
Ex (nm)
Em (nm)
Unit Size
22001Calcein *UltraPure Grade* *CAS 154071-48-4*49551510 mg
22002Calcein, AM *CAS 148504-34-1*4955151 mg
22003Calcein, AM *UltraPure grade* *CAS 148504-34-1*4955151 mg
22004Calcein, AM *UltraPure grade* *CAS 148504-34-1*49551520x50 µg
22006Calcein Blue *CAS 54375-47-2*36044525 mg
22007Calcein Blue, AM *CAS 168482-84-6*3604451 mg
21902Calcein Deep Red™6466591 mg
22010Calcein Deep Red™ acetate6466591 mg
22009Calcein Orange™ diacetate5255501 mg
22008Calcein Orange™ sodium salt5255501 mg
21900Calcein Red™ AM5605741 mg
21901Calcein Red™ sodium salt5605741 mg
21908Calcein UltraBlue™ AM36044510x50 µg
21909Calcein UltraBlue™ sodium salt3604451 mg
22012CytoCalcein™ Violet 450 *Excited at 405 nm*4084501 mg
22013CytoCalcein™ Violet 500 *Excited at 405 nm*4105001 mg