Calcein, AM *UltraPure grade* *CAS 148504-34-1*
| Overview |  SDSProtocol |
CAS 148504-34-1 | Molecular weight 994.86 | Extinction coefficient (cm -1 M -1) 81000 | Excitation (nm) 501 | Emission (nm) 521 |
Calcein AM readily passes through the cell membrane of viable cells because of its enhanced hydrophobicity as compared to calcein. The acetomethoxy (AM) derivate of calcein (calcein AM) is widely used for labeling live cells as it can be transported through the cellular membrane into live cells. The AM ester groups mask the part of the molecule that chelates calcium. Upon transporting into live cells cellular esterases cut off the AM groups, the molecule binds to calcium within cell (resulting in acquiring strong green fluorescence), and gets trapped inside. As dead cells lack esterases, only live cells are marked. This feature makes it very useful for testing of cell viability and for short-term marking of cells. Compared with other live cell-labeling reagents (such as BCECF-AM and carboxy-fluorescein diacetate), calcein-AM is the most suitable fluorescent probe for staining viable cells because of its low cytotoxicity. Calcein does not significantly affect cellular functions such as proliferation or chemotaxis of lymophocyte. In addition, viability assays using calcein are reliable and correlate well with the standard 51Cr-release assay.
Platform
Flow cytometer
| Excitation | 488 nm laser |
| Emission | 530/30 nm filter |
| Instrument specification(s) | FITC channel |
Fluorescence microscope
| Excitation | FITC filter set |
| Emission | FITC filter set |
| Recommended plate | Black wall/clear bottom |
Fluorescence microplate reader
| Excitation | 490 |
| Emission | 525 |
| Cutoff | 515 |
| Recommended plate | Black wall/clear bottom |
| Instrument specification(s) | Bottom read mode |
Example protocol
PREPARATION OF STOCK SOLUTIONS
Unless otherwise noted, all unused stock solutions should be divided into single-use aliquots and stored at -20 °C after preparation. Avoid repeated freeze-thaw cycles.
Note The nonionic detergent Pluronic® F-127 can be used to increase the aqueous solubility of AM esters. In the staining buffer, the final Pluronic® F-127 concentration should be approximately 0.02%. A variety of Pluronic® F-127 products can be purchased from AAT Bioquest. Avoid long-term storage of AM esters in the presence of Pluronic® F-127.
Calcein, AM *UltraPure grade* stock solution
Prepare a 2 to 5 mM stock solution of Calcein AM in high-quality, anhydrous DMSO.Note The nonionic detergent Pluronic® F-127 can be used to increase the aqueous solubility of AM esters. In the staining buffer, the final Pluronic® F-127 concentration should be approximately 0.02%. A variety of Pluronic® F-127 products can be purchased from AAT Bioquest. Avoid long-term storage of AM esters in the presence of Pluronic® F-127.
PREPARATION OF WORKING SOLUTION
Calcein, AM *UltraPure grade* working solution
Prepare a Calcein AM working solution of 1 to 10 µM in the buffer of your choice (e.g., Hanks and Hepes buffer). For most cell lines, Calcein AM at the final concentration of 4 to 5 µM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.Note If your cells contain organic anion-transporters, probenecid (1–2.5 mM) or sulfinpyrazone (0.1–0.25 mM) may be added to the working solution to reduce leakage of the de-esterified indicators.
SAMPLE EXPERIMENTAL PROTOCOL
- Prepare cells for imaging.
- Remove the cell culture medium and wash cells once with serum-free buffer to remove any remaining media.
Note Serum in cell culture media may contain esterase activity, which can increase background interference. - Add Calcein AM working solution to the culture.
- Incubate cells at 37 °C for 30 to 60 minutes.
- Replace the dye working solution with HHBS or buffer of your choice (containing an anion transporter inhibitor, such as 1 mM probenecid, if applicable) to remove any excess probes.
- Measure the fluorescence intensity using either a fluorescence microscope equipped with a FITC filter set, a flow cytometer equipped with a blue laser and a 530/30 nm filter (FITC channel), or a fluorescence plate reader at Ex/Em = 490/525 nm cutoff 515 nm.
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
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Spectrum
Open in Advanced Spectrum Viewer
Spectral properties
| Extinction coefficient (cm -1 M -1) | 81000 |
| Excitation (nm) | 501 |
| Emission (nm) | 521 |
Images
Figure 1. Simultaneous imaging of live and apoptotic HeLa cells labeled using calcein AM (Cat No. 22003) and Annexin V-iFluor® 647 conjugate (Cat No. 20074)
Figure 2. Images of Live HeLa cells stained with Calcein, AM (Cat.22003).
Figure 3. Chemical structure for Calcein, AM *UltraPure grade* *CAS 148504-34-1*
Citations
View all 31 citations: Citation Explorer
Effervescent Atomizer as Novel Cell Spray Technology to Decrease the Gas-to-Liquid Ratio
Authors: Thiebes, Anja Lena and Klein, Sarah and Zingsheim, Jonas and M{\"o}ller, Georg H and G{\"u}rzing, Stefanie and Reddemann, Manuel A and Behbahani, Mehdi and Jockenhoevel, Stefan and Cornelissen, Christian G
Journal: Pharmaceutics (2022): 2421
Authors: Thiebes, Anja Lena and Klein, Sarah and Zingsheim, Jonas and M{\"o}ller, Georg H and G{\"u}rzing, Stefanie and Reddemann, Manuel A and Behbahani, Mehdi and Jockenhoevel, Stefan and Cornelissen, Christian G
Journal: Pharmaceutics (2022): 2421
Sensing of Physical Factors by Cells: Electric Field, Mechanical Forces, Physical Plasma and Light—Importance for Tissue Regeneration
Authors: Bielfeldt, Meike and Rebl, Henrike and Peters, Kirsten and Sridharan, Kaarthik and Staehlke, Susanne and Nebe, J Barbara
Journal: Biomedical Materials \& Devices (2022): 1--16
Authors: Bielfeldt, Meike and Rebl, Henrike and Peters, Kirsten and Sridharan, Kaarthik and Staehlke, Susanne and Nebe, J Barbara
Journal: Biomedical Materials \& Devices (2022): 1--16
Genetic Engineering of Talaromyces marneffei to Enhance Siderophore Production and Preliminary Testing for Medical Application Potential
Authors: Amsri, Artid and Srichairatanakool, Somdet and Teerawutgulrag, Aphiwat and Youngchim, Sirida and Pongpom, Monsicha
Journal: Journal of Fungi (2022): 1183
Authors: Amsri, Artid and Srichairatanakool, Somdet and Teerawutgulrag, Aphiwat and Youngchim, Sirida and Pongpom, Monsicha
Journal: Journal of Fungi (2022): 1183
Drug/bioactive eluting chitosan composite foams for osteochondral tissue engineering
Authors: Samie, Muhammad and Khan, Ather Farooq and Rahman, Saeed Ur and Iqbal, Haffsah and Yameen, Muhammad Arfat and Chaudhry, Aqif Anwar and Galeb, Hanaa A and Halcovitch, Nathan R and Hardy, John G
Journal: International Journal of Biological Macromolecules (2022)
Authors: Samie, Muhammad and Khan, Ather Farooq and Rahman, Saeed Ur and Iqbal, Haffsah and Yameen, Muhammad Arfat and Chaudhry, Aqif Anwar and Galeb, Hanaa A and Halcovitch, Nathan R and Hardy, John G
Journal: International Journal of Biological Macromolecules (2022)
The effects of acute exercise and inflammation on immune function in early-stage prostate cancer
Authors: Schauer, Tim and Djurhuus, Sissal Sigmundsd{\'o}ttir and Simonsen, Casper and Brasso, Klaus and Christensen, Jesper Frank
Journal: Brain, behavior, \& immunity-health (2022): 100508
Authors: Schauer, Tim and Djurhuus, Sissal Sigmundsd{\'o}ttir and Simonsen, Casper and Brasso, Klaus and Christensen, Jesper Frank
Journal: Brain, behavior, \& immunity-health (2022): 100508
Highly Concentrated Nitrogen-Doped Carbon Nanotubes in Alginate--Gelatin 3D Hydrogels Enable in Vitro Breast Cancer Spheroid Formation
Authors: Munguia-Lopez, Jose G and Jiang, Tao and Ferlatte, Audrey and Fajardo-Diaz, Juan L and Munoz-Sandoval, Emilio and Tran, Simon D and Kinsella, Joseph M
Journal: Advanced NanoBiomed Research (2021): 2100104
Authors: Munguia-Lopez, Jose G and Jiang, Tao and Ferlatte, Audrey and Fajardo-Diaz, Juan L and Munoz-Sandoval, Emilio and Tran, Simon D and Kinsella, Joseph M
Journal: Advanced NanoBiomed Research (2021): 2100104
CORRELATION BETWEEN CELL SURVIVAL AND ATOMIZATION CHARACTERISTICS IN AIR-ASSISTED BRONCHOSCOPIC SPRAYING
Authors: G{\"u}rzing, Stefanie and Klein, Sarah and M{\"o}ller, Georg and Thiebes, Anja Lena and Cornelissen, Christian Gabriel and Reddemann, Manuel Armin
Journal: Atomization and Sprays (2021)
Authors: G{\"u}rzing, Stefanie and Klein, Sarah and M{\"o}ller, Georg and Thiebes, Anja Lena and Cornelissen, Christian Gabriel and Reddemann, Manuel Armin
Journal: Atomization and Sprays (2021)
Endoscopic atomization of mesenchymal stromal cells: in vitro study for local cell therapy of the lungs
Authors: Thiebes, Anja Lena and Uhl, Franziska E and Hauser, Marie and Cornelissen, Christian G and Jockenhoevel, Stefan and Weiss, Daniel J
Journal: Cytotherapy (2021): 293--300
Authors: Thiebes, Anja Lena and Uhl, Franziska E and Hauser, Marie and Cornelissen, Christian G and Jockenhoevel, Stefan and Weiss, Daniel J
Journal: Cytotherapy (2021): 293--300
A necroptotic-independent function of MLKL in regulating endothelial cell adhesion molecule expression
Authors: Dai, Jialin and Zhang, Chonghe and Guo, Lin and He, Hao and Jiang, Kai and Huang, Yingying and Zhang, Xixi and Zhang, Haibing and Wei, Wu and Zhang, Yaoyang and others,
Journal: Cell Death \& Disease (2020): 1--16
Authors: Dai, Jialin and Zhang, Chonghe and Guo, Lin and He, Hao and Jiang, Kai and Huang, Yingying and Zhang, Xixi and Zhang, Haibing and Wei, Wu and Zhang, Yaoyang and others,
Journal: Cell Death \& Disease (2020): 1--16
Preparation of phospholipid-based polycarbonate urethanes for potential applications of blood-contacting implants
Authors: Li, Peichuang and Cai, Wanhao and Li, Xin and Wang, Kebing and Zhou, Lei and You, Tianxue and Wang, Rui and Chen, Hang and Zhao, Yuancong and Wang, Jin and others,
Journal: Regenerative biomaterials (2020): 491--504
Authors: Li, Peichuang and Cai, Wanhao and Li, Xin and Wang, Kebing and Zhou, Lei and You, Tianxue and Wang, Rui and Chen, Hang and Zhao, Yuancong and Wang, Jin and others,
Journal: Regenerative biomaterials (2020): 491--504
References
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Localized functional chemical stimulation of TE 671 cells cultured on nanoporous membrane by calcein and acetylcholine
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A vaccination and challenge model using calcein marked fish
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Novel fluorescence assay using calcein-AM for the determination of human erythrocyte viability and aging
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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
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Comparison of the usefulness of the MTT, ATP, and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines
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In vitro assay of mineralized-tissue formation on titanium using fluorescent staining with calcein blue
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The effects of calcium chloride and sodium chloride on the electroporation-mediated skin permeation of fluorescein isothiocyanate (FITC)-dextrans in vitro
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Journal: Biol Pharm Bull (2003): 1508
Authors: Tokudome Y, Sugibayashi K.
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Application notes
Multiplexing Cell Proliferation and Cytotoxicity Assays Using Calcein Red and CytoCalceins
Multiplexing Cell Proliferation and Cytotoxicity Assays Using Calcein Red and CytoCalceins
Multiplexing Cell Proliferation and Cytotoxicity Assays Using Calcein Red and CytoCalceins
Annexin V
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