Calcein AM is a membrane-permeant, fluorogenic esterase substrate commonly used to determine cell viability
and plasma membrane integrity. As a nearly neutral substrate, calcein AM can diffuse freely and noninvasively across the plasma membrane and permeate the cytosol of live cells. Once inside, cytosolic esterases in metabolically active cells rapidly hydrolyze the non-fluorescent calcein AM substrates into calcein products retained by cells with intact plasma membranes. Since dead cells lack esterase activity, only viable cells are labeled. Under blue light excitation, calcein emits a robust green fluorescence proportional to the activity of cellular esterases and the number of viable cells present in the sample. Compared to other live-cell labeling reagents, such as BCECF AM or carboxyfluorescein diacetate, the brighter fluorescence, photostability, and low cytotoxicity of calcein AM is advantageous for a variety of studies, including cell tracking, cell adhesion, chemotaxis,multidrug resistance, cell viability, apoptosis
, and cytotoxicity.
AAT Bioquest offers a wide selection of calcein AM products for cell viability analysis and calcein sodium salt products for utility as reference dyes. In addition to the traditional green fluorescent calcein AM, we provide calcein AM derivatives that emit in blue, orange, red, and deep red fluorescence to accommodate multiplex analysis with GFP-transfected cells and other fluorescent indicators as well as two violet-light excitable CytoCalcein™ dyes for use in flow cytometry.
Calcein Blue AM, Calcein Orange™ Diacetate, Calcein Red™ AM, and Calcein Deep Red™ AM for Multi-Functional Analysis
Calcein AM (22004
) is recognized as the principal indicator of cell viability because of its passive intracellular diffusion, low cytotoxicity, superior cell retention, and strong fluorescence intensity in the physiological pH range. Consequently, the fluorescence emission maximum of calcein at ~521 nm makes it challenging to use in combination with GFP-transfected cells or other green fluorescent indicators for multiplex analysis. To address the single color limitation of calcein AM, AAT Bioquest has developed calcein derivatives in a broad range of color options, including Calcein Blue AM, Calcein Orange™ AM, Calcein Red™ AM, and Calcein Deep Red™ AM dyes for multicolor labeling and functional analysis of live cells (Table 1).
Calcein Blue AM
Calcein Blue AM (22007
) is a UV-light excitable viability indicator that emits bright blue fluorescence (Em = ~441) at pH 4-11 and is designed for use with instruments optimized for detecting blue fluorescence. It can be used in combination with the phosphatidylserine stain Annexin V iFluor® 488 (20071
) and nucleic acid dead cell stain 7-AAD (17501
) for the three-color discrimination of live/apoptotic/necrotic cell populations (Figure 1), as well as with GFP, RFP, and other green- or red-fluorescent probes for multicolor functional analysis.
Calcein Orange™ Diacetate
Calcein Orange™ diacetate (22007
) is blue/green light excitable viability indicator that emits bright orange fluorescence (Em = ~545 nm). Like calcein AM, Calcein Orange™ diacetate is a non-fluorescent membrane-permeant substrate that requires esterase hydrolysis to activate its fluorescence. The excellent cellular retention of Calcein Orange™ by live cells with uncompromised plasma membranes makes it a valuable cell tracer and live-cell labeling reagent. It can be used in combination with blue- or green-fluorescent probes, such as the nucleic acid stain Hoechst 33342 (17535
, Figure 2) or the calcium indicator Cal-520®
Calcein Red™ AM and Calcein Deep Red™ AM
Also available are Calcein Red™ AM (21900
) and Calcein Deep Red™ AM (22011
). Upon hydrolysis by cytosolic esterases, Calcein Red™ (Ex/Em = 562/576 nm) and Calcein Deep Red™ (Ex/Em = 643/663 nm) are well-retained by viable cells with uncompromised membranes and emit either yellow or red fluorescence, respectively, when excited by the appropriate light source. Both Calcein Red™ AM and Calcein Deep Red™ AM can be used in combination with blue- and green-fluorescent probes.
Calcein Orange™ Diacetate
Staurosporine treated Jurkat cells stained using Calcein™ Blue AM (blue,22007
), Annexin V iFluor® 488 (green, 20071
), and 7-AAD (red, 17501
).;Live HeLa cells stained using viability indicator Calcein Orange™ diacetate (22007
) and nucleic acid stain Hoechst 33342 (17535
).;Flow cytometric analysis of live Jurkat cells stained with 2 µM Calcein Deep Red™ AM (22011
, red) and dead cell populations treated in a 55°C water bath for 30 minutes (green) using a NovoCyte 3000 flow cytometer.
Table 1. Properties of calcein AM viability dyes
|Calcein Blue, AM *CAS 168482-84-6*||465.41||354/441 nm||1 mg||22007|
|Calcein, AM *CAS 148504-34-1*||994.86||501/521 nm||1 mg||22002|
|Calcein, AM *UltraPure grade* *CAS 148504-34-1*||994.86||501/521 nm||1 mg||22003|
|Calcein, AM *UltraPure grade* *CAS 148504-34-1*||994.86||501/521 nm||20x50 µg||22004|
|Calcein Orange™ diacetate||877.24||531/545 nm||1 mg||22009|
|Calcein Red™ AM||1015.79||562/576 nm||1 mg||21900|
|Calcein Deep Red™ AM ester||373.66||643/663 nm||1 mg||22011|
CytoCalcein™ Violet Viability Dyes Optimized for Flow Cytometry and Imaging
CytoCalcein™ Violet 450 and CytoCalcein™ Violet 500 are optimized for use in flow cytometry. The enzymatic conversion of non-fluorescent CytoCalcein™ Violet 450 AM and CytoCalcein™ Violet 500 AM to highly fluorescent CytoCalcein™ Violet 450 and CytoCalcein™ Violet 500 products (Ex/Em maxima ~406/445 nm an found in most flow cytometers. In conjunction with red fluorescent nucleic acid dead cell stains propidium iodide (PI, Ex/Em maxima ~537/618 nm) or 7-Aminoactinomycin D (7-AAD, Ex/Em ~549/648 nm), CytoCalcein™ Violet dyes are excellent fluorophores for two-color discrimination of live from dead cell populations based on membrane integrity and esterase activity. Removing data points representing dead cells is a critical step for improving the accuracy of your flow cytometry analysis. Often dead cells will bind non-specifically to many reagents contributing to false-positive results.
CytoCalcein™ Violet 450 and CytoCalcein™ Violet 500 are available as standalone reagents in 1 mg unit size. In addition, CytoCalcein™ Violet 450 serves as the viability indicator in our Cell Meter™ Cell Viability Assay Kit *Blue Fluorescence with 405 nm excitation* (22784
). This kit provides an optimized cell labeling protocol and sufficient reagents, including CytoCalcein™ Violet 450, DMSO, and an assay buffer, for 500 tests using a 200 µL assay volume and a standard fluorescence microplate reader. It can also be used to label viable cells for fluorescence flow cytometry and microscopic investigations of cellular functions.
Excitation and emission spectrum of viability dye CytoCalcein™ Violet 450 (22012
).;Excitation and emission spectrum of viability dye CytoCalcein™ Violet 500 (22013
Table 2. Properties of cytocalcein AM viability dyes optimized for flow cytometry
|CytoCalcein™ Violet 450 *Excited at 405 nm*||∼600||406/445 nm||1 mg||22012|
|CytoCalcein™ Violet 500 *Excited at 405 nm*||517.93||420/505 nm||1 mg||22013|