AAT Bioquest

Cal-520™, the Best Green Fluorescent Ca2+ Dye

Cal-520™ provides the most 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 background ratio and intracellular retention compared to the existing green calcium indicators (such as Fluo-3 AM and Fluo-4 AM). The higher signal/background 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.

Our preliminary in-house research indicated that Cal-520™ AM can be readily loaded to a guinea pig heart and stays there for a few hours in the absence of probenecid. The calcium signal can be readily monitored with Cal-520™ AM while it is difficult to observe the calcium signal under the same conditions with other green calcium dyes such as Fluo-3 AM and Fluo-4 AM.

Key Features of Cal-520™ AM

  • Cal-520™ AM is better retained in live cells than Fluo-3 AM and Fluo-4 AM
  • Cal-520™ AM has much higher signal/background ratio than Fluo-3 AM and Fluo-4 AM in cells
  • Cal-520™ AM has almost identical spectra to those of Fluo-4 AM

Fluo-4 am
Cal 520 am

Responses of endogenous P2Y receptor to ATP in CHO-M1 cells without probenecid. CHO-M1 cells were seeded overnight at 40,000 cells per 100 µL per well in a Costar 96-well black wall/clear bottom plate. 100 µL of 4 µM Fluo-4 AM (left), Cal 520™ AM (right) in HHBS was added into the wells, and the cells were incubated at 37 C for 2 hours. The dye loading medium was replaced with 100 µL HHBS and 50 µL of 300 µM ATP were added. The cells were imaged with a fluorescence microscope (Olympus IX71) using FITC channel.

with probenecid
without probenecid

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 2.5 mm probenecid) was added into the cells, and the cells were incubated at 37°C for 2 hours. ATP (50 µL/well) was added using FlexStationR to achieve the final indicated concentrations.


Table 1. Spectral Comparison of Fluo-3, Fluo-4, Fluo-8® and Cal-520

Ex (nm)
Em (nm)
Kd (nM)
Cal-520™ 4925140.75320
Fluo-3 5065250.15390
Fluo-4 4935150.16345
  • *QY = Fluorescence Quantum Yield in the presence of 5 mM calcium citrate.


Table 2. Spectral and Ca2+-Binding Properties of Cal-520™ and Fluo-8® Calcium Indicators

Ca2+ Indicator
Kd (Ca2+-Binding)
Cal-520™ 492 nm514 nm320 nM
Cal-520FF™ 492 nm 490 nm 9.8 µM
Fluo-8® 490 nm 514 nm 389 nM
Fluo-8H™ 490 nm 490 nm 232 nM
Fluo-8L™ 490 nm 490 nm 1.86 µM
Fluo-8FF™ 490 nm 490 nm 10 µM


Recent Publication Highlights of Cal-520™ AM

  1. M. Tada, A. Takeuchi, M. Hashizume, K. Kitamura and M. Kano. A highly sensitive fluorescent indicator dye for calcium imaging of neural activity in vitro and in vivo. Eur J Neurosci, Published on January 9, 2014 [DOI: 10.1111/ejn.12476]
    1. Tada et al. used Cal-520 AM calcium imaging to monitor the activities of individual neurons in vitro and in vivo. Cal520 AM or Oregon Green 488 BAPTA-1 AM was loaded in neurons under the same conditions. Calcium imaging was performed for more than 30 min after dye injection. Cal-520 AM is sufficiently sensitive to reliably detect single action potentials (APs) both in vitro and in vivo. In neocortical neurons, Cal-520 calcium signals were linearly correlated with the number of APs, and the SNR was > 6 for in vitro slice preparations and > 1.6 for in vivo anesthetised mice. In cerebellar Purkinje cells, dendritic calcium transients evoked by climbing fiber inputs were clearly observed in anesthetized mice with a high SNR and fast decay time. Cal-520 AM demonstrated great advantages over Oregon Green BAPTA-1 AM, the most commonly used calcium indicator dye, for monitoring the activity of individual neurons both in vitro and in vivo.
  2. D.Kodama and A. Togari. Store-operated calcium entry induced by activation of Gq-coupled alpha1B adrenergic receptor in human osteoblast Biochem Biophys Res Comm. 2013, 437(2), 239"244 [doi:10.1016/j.bbrc.2013.06.047]
    1. Kodama and Togari used Cal-520 AM to study the signal transduction pathway of noradrenaline (NA)-induced [Ca2+]i elevation in human osteoblast SaM-1 cells. Cal-520 AM was loaded in human osteoblast SaM-1 cells for 30 min with Hanks and 10 mM HEPES buffer (pH 7.4). The fluorescence of Cal-520 was recorded every 2 seconds with 488 nm excitation, and the data were analyzed with ZEN 2009. The authors demonstrated that the intracellular [Ca2+]i was increased by NA via α1B-AR. The signal pathway of NA-induced [Ca2+]i elevation was monitored using Ca2+ fluorescence imaging in SaM-1 cells. The authors concluded the [Ca2+]i elevation was mediated by Gq protein-coupled α1B-AR, and Ca2+ influx is the predominant pathway of NA-induced [Ca2+]i elevation. Ca2+ influx through store-operated Ca2+ channels plays a critical role in the signal transduction pathway of Gq protein-coupled α1B-AR in human osteoblasts.
  3. R. Yamamoto, S. Ueki, Y. Moritoki, Y. Kobayashi, H. Oyamada,Y. Konno, M. Tamaki, M. Itoga, M. Takeda, W. Ito and J. Chihara. Adiponectin attenuates human eosinophil adhesion and chemotaxis: implications in allergic inflammation. J Asthma 2013, 50(8), 828-35 [doi:10.3109/02770903.2013.816 725]
    1. Yamamoto et al used Cal-520 AM to monitor the expression of eotaxin receptor CCR3 and intracellular calcium influx by flow cytometry. AdipoR1 and AdipoR2 were expressed in human eosinophils. Adiponectin did not affect eosinophil survival or CCR3 expression while eotaxin-enhanced adhesion was inhibited by pretreatment with adiponectin. Adiponectin also diminished eotaxin-directed chemotactic responses by disturbing both velocity and directionality. Calcium influx in response to eotaxin was attenuated by adiponectin. The authors concluded that adiponectin attenuates the eosinophil functions induced by eotaxin without affecting cell viability. The inhibitory effect was associated with diminished calcium signaling rather than altering of surface receptor expression.
  4. M. Liu, J. Liu; J. Liao, Z. Diwu. A Functional Analysis of GPCR and Calcium Channel Targets Using Cal 520 AM Ester. Biophys J, 2012, 102(3), 309a-310a [doi:10.1016/j.bpj.2011.11.1706].
    1. Liu et al evaluated the signal intensity and signal to background ratio of Cal 520 AM in several cell lines including HEK-293, CHO-M1 and Jurkat cells with different receptor signaling pathways. Cal 520 AM were demonstrated to have much better cell retention ability in addition to its significantly higher signal to background ratio comparing to Fluo-3 AM and Fluo-4 AM. Cal-520 AM requires minimal amount of probenecid present while Fluo-3 AM and Fluo-4 AM requires a significantly amount of probenecid present to prevent the dyes from leaking out of host cells. This feature enables Cal-520 AM for calcium assays in probenecid-sensitive cell lines. Cal-520 AM was a significantly improved fluorescent indicator for monitoring intracellular calcium. The high signal-to-noise ratio and good intracellular retention properties make the Cal-520 AM a robust tool for evaluating GPCR and calcium channel targets as well as for screening their agonists and antagonists.