Calbryte™ 520 AM
Ordering information
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Additional ordering information
Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
Quotation | Request |
International | See distributors |
Shipping | Standard overnight for United States, inquire for international |
Physical properties
Dissociation constant (Kd, nM) | 1200 |
Molecular weight | 1090.90 |
Solvent | DMSO |
Spectral properties
Excitation (nm) | 493 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
Storage, safety and handling
Certificate of Origin | Download PDF |
H-phrase | H303, H313, H333 |
Hazard symbol | XN |
Intended use | Research Use Only (RUO) |
R-phrase | R20, R21, R22 |
Storage | Freeze (< -15 °C); Minimize light exposure |
UNSPSC | 12352200 |
Related products
Overview | ![]() ![]() |
Molecular weight 1090.90 | Dissociation constant (Kd, nM) 1200 | Excitation (nm) 493 | Emission (nm) 515 | Quantum yield 0.751 |
The intracellular calcium flux assay is a widely used method in monitoring signal transduction pathways and high throughput screening of G protein"coupled receptors (GPCRs) and calcium channel targets. Followed by Fluo-3 being introduced in 1989, Fluo-4, Fluo-8 and Cal-520 were later developed with improved signal/background ratio, and became the widely used Ca2+ indicators for confocal microscopy, flow cytometry and high throughput screening applications. However, there are still a few severe problems with Fluo-4. For example, as for Fluo-3, in all most all the intracellular calcium assays with Fluo-4 AM, probenecid is required to prevent the cell-loaded Fluo-4 from leaking out of cells. The use of probenecid with Fluo-4-based calcium assays compromises the assay results since probenecid is well-documented to have a variety of complicated cellular effects. Calbryte™ 520, AM is a novel fluorescent and cell-permeable indicator for the measurement of intracellular calcium. Like other dye AM esters, Calbryte™ 520 AM is non-fluorescent and non-activatable. Once Calbryte™ 520 AM enters the cell, it is readily hydrolyzed by intracellular esterase where it becomes activated and responsive to calcium. The activated indicator is now a polar molecule that is incapable of freely diffusing through the cell membrane, essentially trapping it inside the cell. Upon binding calcium ions, Calbryte™ 520 produces a bright fluorescence signal with extremely high signal/background ratio. It has the identical excitation and emission wavelength as Fluo-4, thus the same Fluo-4 assay settings can be readily applied to Calbryte™ 520-based calcium assays. Its greatly improved signal/background ratio and intracellular retention properties make Calbryte™ 520 AM the most robust indicator for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists in live cells.
Platform
Flow cytometer
Excitation | 488 nm laser |
Emission | 530/30 nm filter |
Instrument specification(s) | FITC channel |
Fluorescence microscope
Excitation | FITC |
Emission | FITC |
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/Programmable liquid handling |
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.
Calbryte™ 520 AM Stock Solution
Prepare a 2 to 5 mM stock solution of Calbryte™ 520 AM in anhydrous DMSO.PREPARATION OF WORKING SOLUTION
Calbryte™ 520 AM 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 dye working solution of 2 to 20 µM 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 to 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 solution, 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 into 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
Table 1. Volume of DMSO needed to reconstitute specific mass of Calbryte™ 520 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 | 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
Enter any two values (mass, volume, concentration) to calculate the third.
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
Open in Advanced Spectrum Viewer


Spectral properties
Excitation (nm) | 493 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
Product Family
Name | Excitation (nm) | Emission (nm) | Quantum yield |
Calbryte™ 590 AM | 581 | 593 | - |
Calbryte™ 630 AM | 607 | 624 | - |
Calbryte™-520L AM | 493 | 515 | 0.751 |
Calbryte™-520XL AM | 493 | 515 | 0.751 |
Cal-520®, AM | 493 | 515 | 0.751 |
Mag-520™ AM | 506 | 525 | - |
Images

Figure 1. ATP response was measured in CHO-K1 cells using Calbryte™ 520 AM (Cat No. 20653) and Fluo-4, AM (Cat No. 20550). CHO-K1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of either 10 µg/mL Calbryte™ 520 AM in HH Buffer with probenecid or 10 µg/mL Fluo-4, AM in HH Buffer with probenecid was added to the wells and incubated for 45 minutes at 37°C. Both dye loading solutions were removed and replaced with 200 µL HH Buffer/well. ATP (50 µL/well) was added to achieve the final indicated concentration of 10 µM. Images were acquired on a Keyence microscope in the FITC channel.

Figure 2. Carbachol dose-response was measured in CHO-M1 cells with Calbryte™ 520 AM and Fluo-4 AM. CHO-M1 cells were seeded overnight at 50,000 cells/100 µL/well in a 96-well black wall/clear bottom costar plate. 100 µL of 10 µg/ml Calbryte™ 520 AM in HH Buffer or 10 µg/ml Fluo-4 in HH Buffer was added and incubated for 45 minutes at 37°C. Dye loading solution was then removed and replaced with 200 µL HH Buffer/well. Carbachol (50 µL/well) was added by FlexStation 3 to achieve the final indicated concentrations.
Citations
View all 171 citations: Citation Explorer
An airway-to-brain sensory pathway mediates influenza-induced sickness
Authors: Bin, Na-Ryum and Prescott, Sara L and Horio, Nao and Wang, Yandan and Chiu, Isaac M and Liberles, Stephen D
Journal: Nature (2023): 1--8
Authors: Bin, Na-Ryum and Prescott, Sara L and Horio, Nao and Wang, Yandan and Chiu, Isaac M and Liberles, Stephen D
Journal: Nature (2023): 1--8
Deletion of SNX9 alleviates CD8 T cell exhaustion for effective cellular cancer immunotherapy
Authors: Trefny, Marcel P and Kirchhammer, Nicole and Auf der Maur, Priska and Natoli, Marina and Schmid, Dominic and Germann, Markus and Fernandez Rodriguez, Laura and Herzig, Petra and L{\"o}tscher, Jonas and Akrami, Maryam and others,
Journal: Nature Communications (2023): 86
Authors: Trefny, Marcel P and Kirchhammer, Nicole and Auf der Maur, Priska and Natoli, Marina and Schmid, Dominic and Germann, Markus and Fernandez Rodriguez, Laura and Herzig, Petra and L{\"o}tscher, Jonas and Akrami, Maryam and others,
Journal: Nature Communications (2023): 86
Functional characteristics of hub and wave-initiator cells in b cell networks
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Authors: {\v{S}}terk, Marko and Dolen{\v{s}}ek, Jurij and Klemen, Ma{\v{s}}a Skelin and Bombek, Lidija Krizancic and Leitgeb, Eva Paradiz and Kercmar, Jasmina and Perc, Matjaz and Rupnik, Marjan Slak and Stozer, Andraz and Gosak, Marko
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Pulsed Electric Field Ablation of Esophageal Malignancies and Mitigating Damage to Smooth Muscle: An In Vitro Study
Authors: Gudvangen, Emily and Mangalanathan, Uma and Semenov, Iurii and Kiester, Allen S and Keppler, Mark A and Ibey, Bennett L and Bixler, Joel N and Pakhomov, Andrei G
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Authors: Gudvangen, Emily and Mangalanathan, Uma and Semenov, Iurii and Kiester, Allen S and Keppler, Mark A and Ibey, Bennett L and Bixler, Joel N and Pakhomov, Andrei G
Journal: International Journal of Molecular Sciences (2023): 2854
Intimations on the Development of Effective Treatment for the Prevention of Preterm Birth: $\beta$3 Adrenergic Receptor Signaling in the Human Myometrium
Authors: Asif, Hazik
Journal: (2022)
Authors: Asif, Hazik
Journal: (2022)
The influence of spontaneous and visual activity on the development of direction selectivity maps in mouse retina
Authors: Tiriac, Alexandre and Bistrong, Karina and Pitcher, Miah N and Tworig, Joshua M and Feller, Marla B
Journal: Cell reports (2022): 110225
Authors: Tiriac, Alexandre and Bistrong, Karina and Pitcher, Miah N and Tworig, Joshua M and Feller, Marla B
Journal: Cell reports (2022): 110225
Temporal signals drive the emergence of multicellular information networks
Authors: Li, Guanyu and LeFebre, Ryan and Starman, Alia and Chappell, Patrick and Mugler, Andrew and Sun, Bo
Journal: arXiv preprint arXiv:2202.13462 (2022)
Authors: Li, Guanyu and LeFebre, Ryan and Starman, Alia and Chappell, Patrick and Mugler, Andrew and Sun, Bo
Journal: arXiv preprint arXiv:2202.13462 (2022)
Downregulated Calcium-Binding Protein S100A16 and HSP27 in Placenta-Derived Multipotent Cells Induce Functional Astrocyte Differentiation
Authors: Cheng, Yu-Che and Huang, Chi-Jung and Ku, Wei-Chi and Guo, Shu-Lin and Tien, Lu-Tai and Lee, Yih-Jing and Chien, Chih-Cheng
Journal: Stem Cell Reviews and Reports (2022): 1--14
Authors: Cheng, Yu-Che and Huang, Chi-Jung and Ku, Wei-Chi and Guo, Shu-Lin and Tien, Lu-Tai and Lee, Yih-Jing and Chien, Chih-Cheng
Journal: Stem Cell Reviews and Reports (2022): 1--14
iRhom pseudoproteases regulate ER stress-induced cell death through IP3 receptors and BCL-2
Authors: Dulloo, Iqbal and Atakpa-Adaji, Peace and Yeh, Yi-Chun and Levet, Cl{\'e}mence and Muliyil, Sonia and Lu, Fangfang and Taylor, Colin W and Freeman, Matthew
Journal: Nature Communications (2022): 1--18
Authors: Dulloo, Iqbal and Atakpa-Adaji, Peace and Yeh, Yi-Chun and Levet, Cl{\'e}mence and Muliyil, Sonia and Lu, Fangfang and Taylor, Colin W and Freeman, Matthew
Journal: Nature Communications (2022): 1--18
pH-Dependence of Glucose-Dependent Activity of Beta Cell Networks in Acute Mouse Pancreatic Tissue Slice
Authors: Postic, S and Gosak, M and Tsai, WH and Pfabe, J and Sarikas, S and Stozˇer, A and Korosˇak, D and Yang, SB and Slak Rupnik, M
Journal: Front. Endocrinol. 13: 916688. doi: 10.3389/fendo (2022)
Authors: Postic, S and Gosak, M and Tsai, WH and Pfabe, J and Sarikas, S and Stozˇer, A and Korosˇak, D and Yang, SB and Slak Rupnik, M
Journal: Front. Endocrinol. 13: 916688. doi: 10.3389/fendo (2022)
References
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Obtaining spontaneously beating cardiomyocyte-like cells from adipose-derived stromal vascular fractions cultured on enzyme-crosslinked gelatin hydrogels
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Application notes
FAQ
Are there any calcium indicators that don't require probenecid (PBC)?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?
What is a calcium flux assay?
What is a calcium ion?
Can I intracellularly measure mitochondria calcium flux and changes in mitochondria membrane potential at the same time?
Do you offer any products for measuring intracellular calcium concentration or movement by flow cytometry?
What is a calcium flux assay?
What is a calcium ion?