AAT Bioquest

Cal-520®, AM

ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-520™ AM (red curve), or Fluo-4 AM (blue curve) respectively with (left) or without probenecid (right) under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well plate. 100 µL of 5 µM Fluo-4 AM or Cal 520™ AM in HHBS (with or without probenecid) was added into the cells, and the cells were incubated at 37 °C for 1 hour. ATP (50 μL/well) was added using FlexSation to achieve the final indicated concentrations.
ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-520™ AM (red curve), or Fluo-4 AM (blue curve) respectively with (left) or without probenecid (right) under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well plate. 100 µL of 5 µM Fluo-4 AM or Cal 520™ AM in HHBS (with or without probenecid) was added into the cells, and the cells were incubated at 37 °C for 1 hour. ATP (50 μL/well) was added using FlexSation to achieve the final indicated concentrations.
ATP-stimulated calcium responses of endogenous P2Y receptor in CHO-K1 cells incubated with Cal-520™ AM (red curve), or Fluo-4 AM (blue curve) respectively with (left) or without probenecid (right) under the same conditions. CHO-K1 cells were seeded overnight at 50,000 cells per 100 µL per well in a Costar black wall/clear bottom 96-well plate. 100 µL of 5 µM Fluo-4 AM or Cal 520™ AM in HHBS (with or without probenecid) was added into the cells, and the cells were incubated at 37 °C for 1 hour. ATP (50 μL/well) was added using FlexSation to achieve the final indicated concentrations.
Response of endogenous P2Y receptor to ATP in CHO-K cells. CHO-K cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. 100 µl of 4 µM Cal 520 ™ AM in HHBS with 1 mM probenecid were added into the wells, and the cells were incubated at 37 °C for 1 hour. The dye loading mediums were replaced with 100 µl HHBS and 1 mM probenecid , then imaged with a fluorescence microscope (Olympus IX71) using FITC channel before and after adding 50 µl of 300 µM ATP .
Two-photon calcium responses to tonal stimuli recorded at 140 ms intervals.<strong>&nbsp;</strong>Averaged traces (mean and S.E.M.) of ∆F/F0 in 44 neurons stained with Cal-520 AM. The red trace represents responses to 20 kHz stimuli lasting for 7s, and the blue trace shows responses to 20 kHz stimuli lasting for 1s in the same neurons. The off-responses to stimuli lasting for 7 s were significantly larger than the on-responses to stimuli lasting for 1 s (P&lt;0.0001). Source: <strong>Auditory cortical field coding long-lasting tonal offsets in mice</strong> by Baba et al., <em>Scientific Reports</em>, Sep. 2016.
Functional sperm analysis. (a) Tracks for freely swimming wildtype Prm2+/+ and heterozygous Prm2+/&minus; sperm. (b) Flagellar waveform. Sperm were tethered with their heads to a glass surface and the flagellar waveform was analyzed. One beat cycle was projected. Scale bar: 10&thinsp;&mu;m. (c) Changes in the intracellular Ca<sup>2+</sup> concentration in Prm2+/+, Prm2+/&minus;, and Prm2&minus;/&minus; sperm. Sperm have been loaded with Cal520-AM and stimulated with K8.6 (blue), 10&thinsp;mM 8-Br-cAMP (red), 10&thinsp;mM NH4Cl (green), or 2&thinsp;&mu;M ionomycin (light blue). Experiments have been measured using the stopped-flow technique. (d) Loading of sperm with Cal520-AM. Loading of Prm2+/&minus;, and Prm2&minus;/&minus; sperm was tested using fluorescence microscopy. Scale bar&thinsp;=&thinsp;20&thinsp;&mu;m. Source: <strong>Re-visiting the Protamine-2 locus: deletion, but not haploinsufficiency, renders male mice infertile</strong> by Schneider et al.,&nbsp;<em>Scientific Reports</em>, Nov. 2016.
Selectivity of V1 neurons. A) Neurons stained with Cal-520 but not with SR-101 in the V1 of a wild-type mouse (left) and a Pcdh&alpha;1,12 mouse (right). The image was obtained using a two-photon microscope. B) Sample traces of neuronal calcium responses to moving grating patterns in eight directions (from -45&deg; to 270&deg; in 45&deg; steps) for 2 s in a wild-type mouse (left) and a Pcdh-&alpha;1,12 mouse (right). C) Cumulative distributions of the orientation selectivity index (OSI, left) and direction selectivity index (DSI, right) of neurons obtained from three wild-type mice and three Pcdh-&alpha;1,12 mice. The OSI was obtained from1698 and 1342 neurons, respectively. The DSI was obtained from 365 and 302 neurons with an OSI &gt; 0.45, respectively. There was no significant difference in the cumulative distribution of the OSI or DSI between wild-type and Pcdh&alpha;1,12 mice. Source: <strong>Molecular diversity of clustered protocadherin-&alpha; required for sensory integration and short-term memory in mice </strong>by Yamagishi et al., <em>Scientific Reports</em>, June 2018.
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Physical properties
Dissociation constant (Kd, nM)320
Molecular weight1102.95
Spectral properties
Excitation (nm)493
Emission (nm)515
Quantum yield0.751
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure
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Molecular weight
Dissociation constant (Kd, nM)
Excitation (nm)
Emission (nm)
Quantum yield
Cal-520® AM provides a robust homogeneous fluorescence-based assay tool for detecting intracellular calcium mobilization. Cal-520® AM is a new fluorogenic calcium-sensitive dye with a significantly improved signal to noise ratio and intracellular retention compared to the existing green calcium indicators (such as Fluo-3 AM and Fluo-4 AM). Cells expressing a GPCR or calcium channel of interest that signals through calcium can be preloaded with Cal-520® AM which can cross cell membrane. Once inside the cell, the lipophilic blocking groups of Cal-520™ AM are cleaved by esterases, resulting in a negatively charged fluorescent dye that stays inside cells. Its fluorescence is greatly enhanced upon binding to calcium. When cells stimulated with agonists, the receptor signals the release of intracellular calcium, which significantly increase the fluorescence of Cal-520®. The characteristics of its long wavelength, high sensitivity, and >100 times fluorescence enhancement, make Cal-520® AM an ideal indicator for the measurement of cellular calcium. The high S/N ratio and better intracellular retention make the Cal-520® calcium assay a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists.


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

Cal-520® AM Stock Solution
  1. Prepare a 2 to 5 mM stock solution of Cal-520® AM in high-quality, anhydrous DMSO.

    Note: When reconstituted in DMSO, Cal-520® AM is a clear, colorless solution.


Cal-520® AM Working Solution
  1. On the day of the experiment, either dissolve Cal-520® AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

  2. Prepare a 2 to 20 µM Cal-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, Cal-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 Cal-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.


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 Cal-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.

  3. Incubate the dye-loaded plate in a cell incubator at 37 °C for 1 to 2 hours.

    Note: Incubating the dye for longer than 2 hours 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 Cal-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 mM90.666 µL453.33 µL906.659 µL4.533 mL9.067 mL
5 mM18.133 µL90.666 µL181.332 µL906.659 µL1.813 mL
10 mM9.067 µL45.333 µL90.666 µL453.33 µL906.659 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

Product Family

NameExcitation (nm)Emission (nm)Quantum yield
Cal-520® maleimide4935150.751
Cal-520FF™, AM4935150.751
Cal-520N™, AM4935150.751
Cal-520® amine4935150.751
Cal-520® azide4935150.751
Cal-520® alkyne4935150.751
Cal-590™ AM5745880.621
Cal-630™ AM6096260.371
Calbryte™ 520 AM4935150.751
Cal-500™ AM3884820.481
Mag-520™ AM506525-
SoNa™ 520 AM491511-
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