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Calbryte™ 520 AM
Intracellular calcium flux assays are essential tools for monitoring signal transduction pathways and conducting high-throughput screening of G protein-coupled receptors (GPCRs) and calcium channel targets. Since the introduction of Fluo-3 in 1989, successive generations of calcium indicators—including Fluo-4, Fluo-8, and Cal-520—have delivered progressively improved signal-to-background ratios, establishing themselves as standard indicators for confocal microscopy, flow cytometry, and high-throughput screening applications. Despite its widespread adoption, Fluo-4 presents significant limitations. Most notably, intracellular calcium assays using Fluo-4 AM require probenecid to prevent the dye from leaking out of cells—a requirement shared with its predecessor, Fluo-3. This dependency on probenecid compromises assay reliability, as probenecid exhibits well-documented, complex cellular effects that can confound results.
Calbryte™ 520 AM represents a next-generation solution to these challenges. This novel, cell-permeable calcium indicator enters cells in a non-fluorescent, inactive form. Once inside, intracellular esterases cleave the AM ester, activating the indicator and rendering it calcium-responsive. The hydrolyzed product becomes a polar molecule that cannot cross cell membranes, effectively trapping it within the cell—eliminating the need for probenecid. When bound to calcium ions, Calbryte™ 520 generates an exceptionally bright fluorescent signal with a superior signal-to-background ratio. Importantly, Calbryte™ 520 shares the same excitation and emission wavelengths as Fluo-4, allowing seamless integration into existing Fluo-4 assay protocols. The combination of enhanced signal quality, improved intracellular retention, and probenecid-free operation makes Calbryte™ 520 AM the most robust calcium indicator available for evaluating GPCR and calcium channel targets, as well as screening their agonists and antagonists in living cells.
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
Prepare a 2 to 5 mM stock solution of Calbryte™ 520 AM in anhydrous DMSO.
Note: When reconstituted in DMSO, Calbryte™ 520 AM is a clear, colorless solution.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Calbryte™ 520 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Calbryte™ 520 AM working solution in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Calbryte™ 520 AM at a final concentration of 4-5 μM is recommended. The exact concentration of indicators required for cell loading must be determined empirically.
Note: The nonionic detergent Pluronic® F-127 is sometimes used to increase the aqueous solubility of Calbryte™ 520 AM. A variety of Pluronic® F-127 solutions can be purchased from AAT Bioquest.
Note: If your cells contain organic anion-transporters, probenecid (1-2 mM) may be added to the dye working solution (final in well concentration will be 0.5-1 mM) to reduce leakage of the de-esterified indicators. A variety of ReadiUse™ Probenecid products, including water-soluble, sodium salt, and stabilized solutions, can be purchased from AAT Bioquest.
SAMPLE EXPERIMENTAL PROTOCOL
Following is our recommended protocol for loading AM esters into live cells. This protocol only provides a guideline and should be modified according to your specific needs.
- Prepare cells in growth medium overnight.
On the next day, add 1X Calbryte™ 520 AM working solution to your cell plate.
Note: If your compound(s) interfere with the serum, replace the growth medium with fresh HHBS buffer before dye-loading.
Incubate the dye-loaded plate in a cell incubator at 37 °C for 30 to 60 minutes.
Note: Incubating the dye for longer than 1 hour can improve signal intensities in certain cell lines.
- 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.
- Add the stimulant as desired and simultaneously measure fluorescence using either a fluorescence microscope equipped with a FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at Ex/Em = 490/525 nm cutoff 515 nm.
Calculators
Common stock solution preparation
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 91.667 µL | 458.337 µL | 916.674 µL | 4.583 mL | 9.167 mL |
| 5 mM | 18.333 µL | 91.667 µL | 183.335 µL | 916.674 µL | 1.833 mL |
| 10 mM | 9.167 µL | 45.834 µL | 91.667 µL | 458.337 µL | 916.674 µL |
Molarity calculator
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) | Quantum yield |
| Calbryte™-520L AM | 493 | 515 | 0.751 |
| Calbryte™-520XL AM | 493 | 515 | 0.751 |
| Calbryte™ 590 AM | 581 | 593 | - |
| Calbryte™ 630 AM | 607 | 624 | - |
| Cal-520®, AM | 492 | 515 | 0.751 |
| Mag-520™ AM | 506 | 525 | - |
Citations
Authors: Chan, Wing-Cheung and Zhao, Qian and Wong, Koon Ho and Tang, Hok-Him and Mok, Daniel Kam-Wah and Pardeshi, Lakhansing and Ruan, Ye Chun and Yang, Yang and Wong, Chi-Ming and Wong, Clarence TT and others,
Journal: Nature Communications (2025): 6308
Authors: Ker{\v{c}}mar, Jasmina and Murko, Nastja and Kri{\v{z}}an{\v{c}}i{\'c} Bombek, Lidija and Paradi{\v{z}} Leitgeb, Eva and Pfabe, Johannes and Posti{\'c}, Sandra and Huang, Ya-Chi and Sto{\v{z}}er, Andra{\v{z}} and Koro{\v{s}}ak, Dean and Kozisek, Xaver and others,
Journal: bioRxiv (2025): 2025--03
Authors: McErlain, Tamara and Glass, Morgan J and McCulla, Elizabeth C and Madden, Caitlin A and Ziemer, Lauren E and Overdahl, Kirsten E and Jarmusch, Alan K and Kruhlak, Michael J and Chesler, Alexander and Yang, Howard H and others,
Journal: bioRxiv (2025): 2025--02
Authors: Jia, Hao and Lenz, Martin O and Robinson, Hugh PC
Journal: Biophysical Journal (2025): 492a--493a
Authors: Schickel, Esther and Bender, Tamara and Kaysan, Leon and Hufgard, Simone and Mayer, Margot and Grosshans, David R and Thielemann, Christiane and Schroeder, Insa S
Journal: bioRxiv (2025): 2025--01
References
Authors: Wu, Yanjiao and Xu, Xiaoli and Ma, Lunkun and Yi, Qian and Sun, Weichao and Tang, Liling
Journal: The International Journal of Biochemistry & Cell Biology (2017)
Authors: Lu, Jiang and Yao, Xue-qin and Luo, Xin and Wang, Yu and Chung, Sookja Kim and Tang, He-xin and Cheung, Chi Wai and Wang, Xian-yu and Meng, Chen and Li, Qing and others, undefined
Journal: Neural Regeneration Research (2017): 945
Authors: Yang, Gang and Xiao, Zhenghua and Ren, Xiaomei and Long, Haiyan and Ma, Kunlong and Qian, Hong and Guo, Yingqiang
Journal: Scientific Reports (2017): 41781
Authors: Liu, Jia and Du, Qing and Zhu, He and Li, Yu and Liu, Maodong and Yu, Shoushui and Wang, Shilei
Journal: Int J Clin Exp Med (2017): 6861--6868
Authors: Sun, Xia and Lin, Yi and Huang, Qiansheng and Shi, Junpeng and Qiu, Ling and Kang, Mei and Chen, Yajie and Fang, Chao and Ye, Ting and Dong, Sijun
Journal: Journal of cellular and molecular medicine (2015): 581--594
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