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Calcein Red™ AM
Calcein AM is one of the most popular fluorescent probes used for labeling and monitoring cellular functions of live cells. However, the single color of Calcein AM makes it impossible to use this valuable reagent in the multicolor applications. For example, it is impossible to use Calcein AM in combination of GFP-tranfacted cells due to the same color to GFP. To address this color limitation of Calcein AM, we have developed Calcein Orange™, Calcein Red™ and Calcein Deep Red™. These new Calcein AM analogs enable the multicolor labeling and functional analysis of live cells in combination with Calcein AM. Non-fluorescent Calcein Red™ AM can easily get into live cells and hydrolyzes to generate strongly fluorescent Calcein Red™ dye. Calcein Red™ dye can be monitored with the common TRITC/Cy3 filter set. AAT Bioquest offers Calcein Red™ as a reference dye to Calcein Red™ AM.
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 Red™ AM Stock Solution
Prepare a 2 to 5 mM stock solution of Calcein Red™ 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 Red™ AM Working Solution
Prepare a Calcein Red™ 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 Red™ 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 Red™ 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 TRITC filter set, a flow cytometer equipped with green/yellow laser and a 585/40 nm filter, or a fluorescence plate reader at Ex/Em = 540/590 nm cutoff 570 nm.
Calculators
Common stock solution preparation
Table 1. Volume of DMSO needed to reconstitute specific mass of Calcein Red™ AM 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 | 98.446 µL | 492.228 µL | 984.455 µL | 4.922 mL | 9.845 mL |
| 5 mM | 19.689 µL | 98.446 µL | 196.891 µL | 984.455 µL | 1.969 mL |
| 10 mM | 9.845 µL | 49.223 µL | 98.446 µL | 492.228 µL | 984.455 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) |
| Calcein Deep Red™ | 643 | 663 |
| Calcein UltraBlue™ AM | 359 | 458 |
| Calcein Blue, AM *CAS 168482-84-6* | 354 | 441 |
| Calcein UltraGreen™ AM | 492 | 514 |
| Fura Red, AM *CAS 149732-62-7* | 435 | 639 |
Citations
View all 32 citations: Citation Explorer
Titrating chimeric antigen receptors on CAR T cells enabled by a microfluidic-based dosage-controlled intracellular mRNA delivery platform
Authors: Chen, Yu-Hsi and Mirza, Mahnoor and Jiang, Ruoyu and Lee, Abraham P
Journal: Biomicrofluidics (2024)
Authors: Chen, Yu-Hsi and Mirza, Mahnoor and Jiang, Ruoyu and Lee, Abraham P
Journal: Biomicrofluidics (2024)
Fabrication of endothelialized capillary-like microchannel networks using sacrificial thermoresponsive microfibers
Authors: Rector, John A and McBride, Lucas and Weber, Callie and Grossman, Kira and Sorets, Alexander and Ventura-Antunes, Lissa and Holtz, Isabella Ko and Young, Katherine and Schrag, Matthew and Lippmann, Ethan S and others,
Journal: Biofabrication (2024)
Authors: Rector, John A and McBride, Lucas and Weber, Callie and Grossman, Kira and Sorets, Alexander and Ventura-Antunes, Lissa and Holtz, Isabella Ko and Young, Katherine and Schrag, Matthew and Lippmann, Ethan S and others,
Journal: Biofabrication (2024)
Titering of Chimeric Antigen Receptors on CAR T Cells enabled by a Microfluidic-based Dosage-Controlled Intracellular mRNA Delivery Platform
Authors: Chen, Yu-Hsi and Jiang, Ruoyu and Lee, Abraham P
Journal: bioRxiv (2023): 2023--03
Authors: Chen, Yu-Hsi and Jiang, Ruoyu and Lee, Abraham P
Journal: bioRxiv (2023): 2023--03
High-Throughput Neurite Outgrowth Assay Using GFP-Labeled iPSC-Derived Neurons
Authors: Zhang, Li and Li, Shuaizhang and Xia, Menghang
Journal: Current Protocols (2022): e542
Authors: Zhang, Li and Li, Shuaizhang and Xia, Menghang
Journal: Current Protocols (2022): e542
AMPA receptor and RNA processing gene dysregulation are early determinants of selective motor neuron vulnerability in a mouse model of amyotrophic lateral sclerosis
Authors: Zanganeh, Pardis F and Barton, Samantha K and Lim, Katherine and Qian, Elizabeth L and Crombie, Duncan E and Bye, Christopher R and Turner, Bradley J
Journal: Brain Communications (2022)
Authors: Zanganeh, Pardis F and Barton, Samantha K and Lim, Katherine and Qian, Elizabeth L and Crombie, Duncan E and Bye, Christopher R and Turner, Bradley J
Journal: Brain Communications (2022)
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
Page updated on December 17, 2024
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