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Calcein

What is calcein?


Calcein, also known as fluorexon, fluorescein complex, or oftasceine, belongs to the family of xanthene dyes. It is a bright, green-fluorescent dye with maximum excitation and emission wavelengths at 501 nm and 521 nm, respectively. The fluorescence of calcein self-quenches at concentrations exceeding 70 mM, or it can be quenched by certain cations, such as Co2+, Ni2+, and Cu2+. The latter can be exploited to evaluate mitochondrial permeability transition pore (mPTP) status and changes in cell volume.

Calcein is typically modified with acetoxymethyl esters (e.g., calcein AM) to promote membrane permeability and is used extensively as a viability indicator to detect live cells. It has been associated with several drug-related assays, including cytotoxicity, oxidative function, and neurotoxicity. (7)

 

How does calcein AM work?


Calcein AM, the acetomethoxy derivative of calcein, is a membrane-permeant fluorogenic esterase substrate used to determine cell viability and membrane integrity. The lipophilic nature of calcein AM allows it to diffuse freely across the cell membrane and permeate the cytosol of live cells. Once inside, non-specific intracellular esterases enzymatically remove the AM group, and the resulting de-esterified calcein products get trapped inside the cell. Under blue light excitation, calcein emits bright green fluorescence at 521 nm. The fluorescence intensity of calcein is directly proportional to the activity of cellular esterases and the number of viable cells present in the sample. (1)


Figure 1. Calcein AM hydrolysis Non-specific intracellular esterase cleavage of AM ester groups converts calcein AM into highly fluorescent calcein.

 

Is calcein AM toxic?


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. (8)

Link: Calcein AM SDS sheet

 

Is calcein AM fluorescent?


No, calcein AM is a non-fluorescent esterase substrate. However, in live cells, calcein AM can passively cross the cell membrane and enter the cytosol, whereby non-specific intracellular esterases hydrolyze the acetoxymethyl ester from calcein AM to yield a highly fluorescent calcein product. Calcein has excitation and emission peaks at 501 nm and 521 nm, respectively, and produces an intense green fluorescence upon excitation. The signal intensity measured is directly proportional to the activity of cellular esterases at play and the number of viable cells present. (2)

 

How long does calcein AM last in cells?


Hydrolyzed calcein AM (e.g., calcein) is a hydrophobic 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 (link to image). 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. (6)


Figure 1. 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. (3)


Calcein AM microbial stain
Figure 2. A mixed population of Live/Dead Bacillus subtilis were stained with calcein AM and propidium iodide (Cat No. 17517).

 

Is calcein cell permeable?


No, calcein is not cell permeable. Calcein contains four carboxylate groups which give the compound an overall negative charge, and charged molecules cannot cross the plasma membrane via simple diffusion because the charges are repelled by the hydrophobic tails in the interior of the phospholipid bilayer. To facilitate permeability, calcein is labeled with several acetoxymethyl ester groups. These lipophilic groups, when attached to calcein (e.g., calcein AM), masks the negative charge creating an uncharged hydrophobic compound. This increase in hydrophobicity enables calcein AM to freely diffuse across intact plasma membranes and permeate the cytosol of live cells. (9)

 

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.

(5)

Table 1. Common stock solution preparation. The volume of DMSO needed to reconstitute specific mass of calcein, AM *CAS 148504-34-1* to given concentration. Note that volume is only for preparing stock solutions. Refer to sample experimental protocol for appropriate experimental buffers.

  50 µg 100 µg 500 µg 1 mg 5 mg
1 mM 50.258 µL 100.517 µL 502.583 µL 1.005 mL 5.026 mL
5 mM 10.052 µL 20.103 µL 100.517 µL 201.033 µL 1.005 mL
10 mM 5.026 µL 10.052 µL 50.258 µL 100.517 µL 502.583 µL

 

What is the function of the AM (acetoxymethyl) group on calcein?


The AM ester in calcein AM has two key functions. First, AM esters, when attached to calcein, mask the negatively charged carboxylic groups of calcein and transform them into an uncharged hydrophobic compound. This facilitates cell permeability by enabling calcein AM to freely cross the plasma membrane and permeate the cytosol. Second, calcein dyes, while in AM ester form, are non-fluorescent. Fluorescence is restored once the cells have permeated the cytosol and non-specific intracellular esterases have cleaved off the AM groups. Moreover, hydrolysis of the AM esters restores the negative charge of calcein and traps the dye within the cell. (4)

 

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. (2)

 

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. Since calcein AM substrates not cleaved by intracellular esterases are non-fluorescent, wash steps are not required prior to imaging. (8)


Calcein UltraBlue™ AM live and dead cell stain V imaging
Figure 3. Fluorescence images of HeLa cells stained with Calcein UltraBlue™ AM (Cat No. 21908) in a Costar black wall/clear bottom 96-well plate. Left: Live HeLa cells in HH buffer; Right: Dead/necrotic cells fixed with ice-cold methanol.

 

References


  1. Bauer, B., Miller, D. S., & Fricker, G. (2003). Compound profiling for P-glycoprotein at the blood-brain barrier using a microplate screening system. Pharmaceutical research, 20(8), 1170–1176. https://doi.org/10.1023/a:1025040712857
  2. Bratosin, D., Mitrofan, L., Palii, C., Estaquier, J., & Montreuil, J. (2005). Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 66(1), 78–84. https://doi.org/10.1002/cyto.a.20152
  3. Hiraoka, Y., & Kimbara, K. (2002). Rapid assessment of the physiological status of the polychlorinated biphenyl degrader Comamonas testosteroni TK102 by flow cytometry. Applied and environmental microbiology, 68(4), 2031–2035. https://doi.org/10.1128/AEM.68.4.2031-2035.2002
  4. Kantevari, S., Gordon, G. R., MacVicar, B. A., & Ellis-Davies, G. C. (2011). A practical guide to the synthesis and use of membrane-permeant acetoxymethyl esters of caged inositol polyphosphates. Nature protocols, 6(3), 327–337. https://doi.org/10.1038/nprot.2010.194
  5. Kitchen, P., Salman, M. M., Abir-Awan, M., Al-Jubair, T., Törnroth-Horsefield, S., Conner, A. C., & Bill, R. M. (2020). Calcein Fluorescence Quenching to Measure Plasma Membrane Water Flux in Live Mammalian Cells. STAR protocols, 1(3), 100157. https://doi.org/10.1016/j.xpro.2020.100157
  6. Miles, F. L., Lynch, J. E., & Sikes, R. A. (2015). Cell-based assays using calcein acetoxymethyl ester show variation in fluorescence with treatment conditions. Journal of biological methods, 2(3), e29. https://doi.org/10.14440/jbm.2015.73
  7. National Center for Biotechnology Information (2021). PubChem Compound Summary for CID 390986, Calcein AM. Retrieved December 6, 2021 from https://pubchem.ncbi.nlm.nih.gov/compound/Calcein-AM
  8. Ramirez, C. N., Antczak, C., & Djaballah, H. (2010). Cell viability assessment: toward content-rich platforms. Expert opinion on drug discovery, 5(3), 223–233. https://doi.org/10.1517/17460441003596685
  9. Vidavsky, N., Addadi, S., Schertel, A., Ben-Ezra, D., Shpigel, M., Addadi, L., & Weiner, S. (2016). Calcium transport into the cells of the sea urchin larva in relation to spicule formation. Proceedings of the National Academy of Sciences of the United States of America, 113(45), 12637–12642. https://doi.org/10.1073/pnas.1612017113


Original created on December 3, 2021, last updated on December 3, 2021
Tagged under: calcein, calcein AM, calcein assay, calcein staining, calcein am excitation and emission, calcein am live/dead, Cell Viability, Proliferation & Function