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Calbryte™ 520 AM

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.
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.
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.
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Catalog Number20650
Unit Size
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Additional ordering information
InternationalSee distributors
ShippingStandard overnight for United States, inquire for international
Physical properties
Dissociation constant (Kd, nM)1200
Molecular weight1090.90
Spectral properties
Excitation (nm)493
Emission (nm)515
Quantum yield0.751
Storage, safety and handling
Certificate of OriginDownload PDF
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Dissociation constant (Kd, nM)
Excitation (nm)
Emission (nm)
Quantum yield
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.


Flow cytometer

Excitation488 nm laser
Emission530/30 nm filter
Instrument specification(s)FITC channel

Fluorescence microscope

Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling

Example protocol


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.


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.


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.
  1. Prepare cells in growth medium overnight.
  2. 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.
  3. 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.
  4. 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.
  5. 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. 


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 mg0.5 mg1 mg5 mg10 mg
1 mM91.667 µL458.337 µL916.674 µL4.583 mL9.167 mL
5 mM18.333 µL91.667 µL183.335 µL916.674 µL1.833 mL
10 mM9.167 µL45.834 µL91.667 µL458.337 µL916.674 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles


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Spectral properties

Excitation (nm)493
Emission (nm)515
Quantum yield0.751


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