Cal-520®, AM

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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.
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21131 $295

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Telephone: 1-800-990-8053
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Ex/Em (nm)492/514
Kd (nM)320
Storage Freeze (<-15 °C)
Minimize light exposure
Category Cell Biology
pH and Ion Indicators
Related Calcium
Cal-520® AM is a next-generation fluorescent calcium indicator with an Ex/Em of 492/514 nm. It has several critical advantages and improvements over older generations of fluorescent Ca2+ probes such as Fluo-3 AM and Fluo-4 AM, namely:
  • Enhanced Brightness and Sensitivity
  • Significantly Higher Signal to Noise Ratio
  • More Consistent Loading and Localization
  • Convenient Spectrum and Easy Multiplexing
Cal-520® AM is excellent for studying G protein-coupled receptors (GPCRs) and calcium ion channels. For a sampling of specific applications, please consult the References and Citations section below.

The History

Every single year, there are hundreds of thousands of peer-reviewed papers published focusing on calcium and its role in biological systems. The reason for this is clear. Calcium ions (Ca2+) play a critical part in many biological processes, from the contracting of muscles to the release of neurotransmitters. This pervasiveness of calcium in biology, and the subsequent need to study it, has pushed for the development of viable, convenient calcium indicators, tools that researchers may use to quantify and visualize intracellular calcium.

The first big breakthrough came in the 1980s, when a team of scientists at the University of California Berkeley developed a fluorescein-based calcium indicator. Their research paved the way for now well-established calcium probes such as Fluo-3 AM and Fluo-4 AM. These fluorescein-based AM esters readily pass through the membranes of living cells, where once inside, they are rapidly hydrolyzed by esterases and set to detect calcium. Upon successful binding with calcium, Fluo-3 or Fluo-4 will become fluorescent, emitting a signal at ~520 nm when excited by a ~490 nm light source.

A significant achievement in its own right, these first generation fluorescein-based dyes did however suffer from several major drawbacks. One of these drawbacks was the high background fluorescence, sometimes termed noise. In general, noise creates problems in data analysis and can obscure possibly significant results, and should be minimized as much as possible. That is why we set out to synthesize a new, low noise fluorescent calcium indicator. After much research and optimizing by our dedicated team of experts, we have developed a new high signal to noise ratio calcium indicator, Cal-520® AM.

Cal-520® AM Features

Cal-520® AM is a next-generation fluorescent calcium indicator that has significant improvements over Fluo-3 AM and Fluo-4 AM. First, by improving the binding specificity of Cal-520® for calcium ions, we have managed to produce an indicator which reduces autofluorescence. This simultaneously lowers background fluorescence and raises the signal to noise ratio. The binding of Cal-520® to Ca2+ will result in a greater than one hundred fold increase in fluorescence intensity. Additionally, we have optimized Cal-520®'s chemical structure so that it is better localized. Unlike Rhod-2, which will localize in mitochondria, Cal-520® will predominantly localize in the cytosol. Lastly, we have developed Cal-520® AM for easy use in multiplexing. While Cal-520® AM retains the familiar spectra of Fluo-4 AM and Fluo-8® AM, we also have Cal-590™ AM (Ex/Em = 573/588 nm) and Cal-630™ (Ex/Em = 608/626 nm) for longer wavelength excitations.

Table 1. Various Excitations and Emissions for Multiplex Assays
Cat No.ProductEx (nm)Em (nm)Kd (nM)Additional Notes
21131Cal-520™ AM492514320Best used with 488 nm laser and FITC filter set
20512Cal-590™ AM573588561Best used with 555 nm laser and TRITC/Cy3 filter set
20532Cal-630™ AM608626792Best used with 594 nm laser and Texas Red® filter set

Because of Cal-520® AM's excellent chemical properties, it is a robust probe for fluorescence-based assay detection of intracellular calcium mobilization. It is perfect for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists. Additionally, Cal-520® AM is completely compatible with use in flow cytometry, fluorescence microscopy, fluorescence spectroscopy and the fluorescence microplate platform. For a sampling of specific applications, please consult the References and Citations section below.

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.

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Table 2. Enter any two values (mass, volume, concentration) to calculate the third.

Mass Molecular weight Volume Concentration Moles
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Wavelength (nm)

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Cal-520® AM has been used by researchers across the globe to investigate the role of calcium ions in critical biological processes. Such processes include, but are not limited to, Ca2+ channel activity, G protein-coupled receptor signaling pathways, intracellular/cytosolic Ca2+ flux, calcium bursts, action potentials and activation of cell receptors.

Below, you may find a small sampling of specific Cal-520® AM applications. To inquire about a potential application of Cal-520® AM, or to consult with our fluorescent dye specialists, please contact us at or 1-800-990-8053.

In Neurobiology, Cal-520® AM has been used to study:
» Neuron single action potentials in neocortical neurons both in vitro and in vivo, and associated voltage-dependent calcium channels[1]
» Superior colliculus in mice in conjunction with two-photon calcium imaging to visualize retinal ganglion cells in the superficial lamina[2]
» Aldolase C compartments in mice, looking at complex spike synchrony and its relation to sensory processing in awake animals[3]
» Neural circuits in brain slice and whole brain preparation, specifically looking at individual action potentials in vivo[4]
» Ca2+ dependent cell signaling pathways using cultured human neuroblastoma SH-SY5Y cells and high-speed video-microscopy[5]

In Cell Signaling, Cal-520® AM has been used to study:
» Intracellular calcium in sperm using the microplate reader platform as a means to quantitating parameters such as motility[6]
» Calcium signaling pathways in meniscus fibrochondrocytes by way of visualizing calcium localization and concentration[7]
» Ca2+ mediated cellular signaling in relation to inositol triphosphate and its flux through gap junctions[8]
» Retinal wave-mediated formation of calcium transients in Müller glial cells with focus on expression of GCaMP3[9]
» Ca2+ signaling pathways in zebrafish sperm, specifically looking at calcium flux resulting from cGMP-induced hyperpolarization[10]

In Cardiology, Cal-520® AM has been used to study:
» Sarcoplasmic reticulum insofar as its role in cardiac excitation-contraction coupling and calcium spark events[11]
» Optical mapping of calcium in cardiac tissue slices to develop a framework for future investigations into calcium transients[12]
» Sodium-calcium exchanger functionality and mechanism with regards to burst pacemaker activity in knockout mice[13]
» Pacemaker modulation in embryonic heart as a function of inositol-1,4,5-triphosphate receptors[14]
» Calcium current and the role of potassium channel-interacting protein 2 (KChIP2) in mice with regards to heart failure[15]

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