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Calcein AM
UltraPure grade; CAS 148504-34-1
Calcein-AM is a green fluorogenic dye that stains live cells for cell viability in flow cytometry and fluorescence microscopy. It is the acetoxymethyl (AM) ester form of the fluorescent probe, calcein.
Principle and mechanism
Calcein is a bright green fluorophore that has enhanced fluorescence upon binding intracellular free calcium ions. Comparatively, its AM ester derivative is non-fluorescent and neutrally charged. These two properties are critical to calcein AM as a specific stain for viable cells. First, the neutral charge confers an increase in hydrophobicity, which enables calcein AM to be transported across the membrane of live cells. Second, the AM ester masks the calcium chelation site, which is necessary for fluorescence. Only in live cells, with active intracellular esterases, is the AM ester group be cleaved, calcium allowed to bind and fluorescence emitted. Moreover, the conversion to the hydrophilic calcein molecule traps it within the cell, whereas dead cells do not retain the dye. In this way, calcein AM specifically selects for viable cells.
Excitation and emission
Calcein-AM is a non-fluorescent compound with a molecular weight of 994.86 daltons. It is hydrolyzed by non-specific intracellular estrases into calcein. Calcein is a fluorescent compound with an excitation peak at 501 nm and an emission peak at 521 nm. In flow cytometry, it can be excited by a 488 nm laser and read in the FITC channel. In a fluorescence plate reader, a setting of Ex/Em = 490/525 nm can be used, with a cutoff at 515 nm.
Toxicity
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
Link: Calcein AM SDS sheet
How long does calcein AM last in cells?
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.
Is calcein AM fixable?
Simply stated, NO calcein AM is NOT FIXABLE. Regardless of the fixation method used - aldehyde or methanol fixation - cells stained with calcein AM will lose their staining pattern. Furthermore, calcein AM cannot be used on fixed cells because they lack the necessary esterases to convert calcein AM to calcein and the membrane integrity needed to retain the dye. Calcein AM is a fluorogenic esterase substrate and should only be used on live cells to determine cell viability.
Does calcein AM stain bacteria?
Calcein AM staining is not only well suited for eukaryote samples, it can also be used to determine cell viability in bacterial samples. Calcein AM is fit for detecting many live, gram-negative bacterial strains and can be used for microbiology studies, environmental monitoring, pharmaceutical sterility testing, and food and plant assessment technologies. Like eukaryotic cells, bacteria contain microbial esterases in their cytoplasm that can cleave the lipophilic blocking groups of calcein AM and convert them into fluorescent calcein. The signal can be measured using a fluorescent instrument, and its intensity is proportional to the number of viable bacteria present.
How do you make a calcein solution?
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.
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.
What are calcein AM positive and negative controls?
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.
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.
Does calcein AM stain dead or necrotic cells?
No, calcein AM does not stain dead or necrotic cells because they lack the necessary enzymes (e.g., non-specific intracellular esterases) to convert non-fluorescent calcein AM to fluorescent calcein.
Calcein AM staining protocol
Quick summary
- Prepare stock solution (2 to 5 mM)
- Prepare working solution (1 to 10 µM)
- Prepare cells for imaging
- Remove cell culture medium and wash cells
- Add working solution to culture
- Incubate at 37 °C (30 to 60 minutes)
- Replace working solution to remove excess probes
- Measure fluorescence (Ex/Em = 490/525 nm)
Full protocol
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of Calcein, AM *UltraPure grade* *CAS 148504-34-1* to given concentration. Note that volume is only for preparing stock solution. Refer to sample experimental protocol for appropriate experimental/physiological buffers.
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 100.517 µL | 502.583 µL | 1.005 mL | 5.026 mL | 10.052 mL |
| 5 mM | 20.103 µL | 100.517 µL | 201.033 µL | 1.005 mL | 2.01 mL |
| 10 mM | 10.052 µL | 50.258 µL | 100.517 µL | 502.583 µL | 1.005 mL |
Molarity calculator
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Citations
View all 39 citations: Citation Explorer
Silicon-rhodamine functionalized evocalcet probes (EvoSiR) potently and selectively label calcium sensing receptors (CaSR) in vitro, in vivo and ex vivo
Authors: Batora, Daniel and Fischer, Jerome P and Kaderli, Reto M and Varga, Mate and Lochner, Martin and Gertsch, Jurg
Journal: bioRxiv (2024): 2024--02
Authors: Batora, Daniel and Fischer, Jerome P and Kaderli, Reto M and Varga, Mate and Lochner, Martin and Gertsch, Jurg
Journal: bioRxiv (2024): 2024--02
An ATPase-Mimicking MXene nanozyme pharmacologically breaks the ironclad defense system for ferroptosis cancer therapy
Authors: Wang, Huan and Liu, Xinchen and Yan, Xiangyu and Du, Yong and Pu, Fang and Ren, Jinsong and Qu, Xiaogang
Journal: Biomaterials (2024): 122523
Authors: Wang, Huan and Liu, Xinchen and Yan, Xiangyu and Du, Yong and Pu, Fang and Ren, Jinsong and Qu, Xiaogang
Journal: Biomaterials (2024): 122523
Elucidating the distinctive regulatory effects and mechanisms of active compounds in Salvia miltiorrhiza Bunge via network pharmacology: Unveiling their roles in the modulation of platelet activation and thrombus formation
Authors: Zhang, Ying and Xin, Guang and Zhou, Qilong and Yu, Xiuxian and Feng, Lijuan and Wen, Ao and Zhang, Kun and Wen, Tingyu and Zhou, Xiaoli and Wu, Qiuling and others,
Journal: Toxicology and Applied Pharmacology (2024): 116871
Authors: Zhang, Ying and Xin, Guang and Zhou, Qilong and Yu, Xiuxian and Feng, Lijuan and Wen, Ao and Zhang, Kun and Wen, Tingyu and Zhou, Xiaoli and Wu, Qiuling and others,
Journal: Toxicology and Applied Pharmacology (2024): 116871
Reinforced Immunogenic Endoplasmic Reticulum Stress and Oxidative Stress via an Orchestrated Nanophotoinducer to Boost Cancer Photoimmunotherapy
Authors: Yang, Zhenzhen and Teng, Yulu and Lin, Meng and Peng, Yiwei and Du, Yitian and Sun, Qi and Gao, Datong and Yuan, Quan and Zhou, Yu and Yang, Yiliang and others,
Journal: ACS nano (2024)
Authors: Yang, Zhenzhen and Teng, Yulu and Lin, Meng and Peng, Yiwei and Du, Yitian and Sun, Qi and Gao, Datong and Yuan, Quan and Zhou, Yu and Yang, Yiliang and others,
Journal: ACS nano (2024)
Type I collagen and fibromodulin enhance the tenogenic phenotype of hASCs and their potential for tendon regeneration
Authors: Tu, Tian and Shi, Yuan and Zhou, Boya and Wang, Xiaoyu and Zhang, Wenjie and Zhou, Guangdong and Mo, Xiumei and Wang, Wenbo and Wu, Jinglei and Liu, Wei
Journal: npj Regenerative Medicine (2023): 67
Authors: Tu, Tian and Shi, Yuan and Zhou, Boya and Wang, Xiaoyu and Zhang, Wenjie and Zhou, Guangdong and Mo, Xiumei and Wang, Wenbo and Wu, Jinglei and Liu, Wei
Journal: npj Regenerative Medicine (2023): 67
References
View all 84 references: Citation Explorer
Functional evidence that the self-renewal gene NANOG regulates esophageal squamous cancer development
Authors: Li, Deng and Xiang, Xiaocong and Yang, Fei and Xiao, Dongqin and Liu, Kang and Chen, Zhu and Zhang, Ruolan and Feng, Gang
Journal: Biochemical and Biophysical Research Communications (2017)
Authors: Li, Deng and Xiang, Xiaocong and Yang, Fei and Xiao, Dongqin and Liu, Kang and Chen, Zhu and Zhang, Ruolan and Feng, Gang
Journal: Biochemical and Biophysical Research Communications (2017)
Localized functional chemical stimulation of TE 671 cells cultured on nanoporous membrane by calcein and acetylcholine
Authors: Zibek S, Stett A, Koltay P, Hu M, Zengerle R, Nisch W, Stelzle M.
Journal: Biophys J. (2006)
Authors: Zibek S, Stett A, Koltay P, Hu M, Zengerle R, Nisch W, Stelzle M.
Journal: Biophys J. (2006)
A vaccination and challenge model using calcein marked fish
Authors: Klesius PH, Evans JJ, Shoemaker CA, Pasnik DJ.
Journal: Fish Shellfish Immunol (2006): 20
Authors: Klesius PH, Evans JJ, Shoemaker CA, Pasnik DJ.
Journal: Fish Shellfish Immunol (2006): 20
Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging
Authors: Bratosin D, Mitrofan L, Palii C, Estaquier J, Montreuil J.
Journal: Cytometry A (2005): 78
Authors: Bratosin D, Mitrofan L, Palii C, Estaquier J, Montreuil J.
Journal: Cytometry A (2005): 78
Cytotoxic effects of 100 reference compounds on Hep G2 and HeLa cells and of 60 compounds on ECC-1 and CHO cells. I mechanistic assays on ROS, glutathione depletion and calcein uptake
Authors: Schoonen WG, Westerink WM, de Roos JA, Debiton E.
Journal: Toxicol In Vitro (2005): 505
Authors: Schoonen WG, Westerink WM, de Roos JA, Debiton E.
Journal: Toxicol In Vitro (2005): 505
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