Cal-520FF™, AM
Ordering information
Price | |
Catalog Number | |
Unit Size | |
Quantity |
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) | 9800 |
Molecular weight | 1138.92 |
Solvent | DMSO |
Spectral properties
Excitation (nm) | 493 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
Storage, safety and handling
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 |
Overview | ![]() ![]() |
Molecular weight 1138.92 | Dissociation constant (Kd, nM) 9800 | Excitation (nm) 493 | Emission (nm) 515 | Quantum yield 0.751 |
Cal-520® 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. Compared to other Cal-520® indicators, Cal-520FF™ has the lowest affinity to calcium with Kd ~ 10 uM.
Platform
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.
Cal-520FF™ AM Stock Solution
Prepare a 2 to 5 mM stock solution of Cal-520FF™ AM in high-quality, anhydrous DMSO. PREPARATION OF WORKING SOLUTION
Cal-520FF™ AM Working Solution
On the day of the experiment, either dissolve Cal-520FF™ 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, Cal-520FF™ 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-520FF™ 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 Cal-520FF™ 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 1 to 2 hours.
Note Incubating the dye for longer than 2 hours 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 Cal-520FF™, 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 | 87.802 µL | 439.012 µL | 878.025 µL | 4.39 mL | 8.78 mL |
5 mM | 17.56 µL | 87.802 µL | 175.605 µL | 878.025 µL | 1.756 mL |
10 mM | 8.78 µL | 43.901 µL | 87.802 µL | 439.012 µL | 878.025 µL |
Molarity calculator
Enter any two values (mass, volume, concentration) to calculate the third.
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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 |
Cal-520®, AM | 493 | 515 | 0.751 |
Cal-520N™, AM | 493 | 515 | 0.751 |
Cal-590™ AM | 574 | 588 | 0.621 |
Cal-630™ AM | 609 | 626 | 0.371 |
Cal-500™ AM | 388 | 482 | 0.481 |
Citations
View all 95 citations: Citation Explorer
KS0365, a novel activator of the transient receptor potential vanilloid 3 (TRPV3) channel, accelerates keratinocyte migration
Authors: Maier, Marion and Olthoff, Stefan and Hill, Kerstin and Zosel, Carolin and Magauer, Thomas and Wein, Lukas Anton and Schaefer, Michael
Journal: British Journal of Pharmacology (2022): 5290--5304
Authors: Maier, Marion and Olthoff, Stefan and Hill, Kerstin and Zosel, Carolin and Magauer, Thomas and Wein, Lukas Anton and Schaefer, Michael
Journal: British Journal of Pharmacology (2022): 5290--5304
Cal-520FF is the Present Optimal Ca 2+ Indicator for Ultrafast Ca 2+ Imaging and Optical Measurement of Ca 2+ Currents
Authors: Bl{\"o}mer, Laila Ananda and Filipis, Luiza and Canepari, Marco
Journal: Journal of Fluorescence (2021): 619--623
Authors: Bl{\"o}mer, Laila Ananda and Filipis, Luiza and Canepari, Marco
Journal: Journal of Fluorescence (2021): 619--623
Advances in Two-Photon Scanning and Scanless Microscopy Technologies for Functional Neural Circuit Imaging
Authors: Schultz, Simon R and Copel, undefined and , Caroline S and Foust, Am and a J , undefined and Quicke, Peter and Schuck, Renaud
Journal: Proceedings of the IEEE (2017): 139--157
Authors: Schultz, Simon R and Copel, undefined and , Caroline S and Foust, Am and a J , undefined and Quicke, Peter and Schuck, Renaud
Journal: Proceedings of the IEEE (2017): 139--157
Interstitial cell modulation of pyeloureteric peristalsis in the mouse renal pelvis examined using FIBSEM tomography and calcium indicators
Authors: Hashitani, Hikaru and Nguyen, Michael J and Noda, Haruka and Mitsui, Retsu and Higashi, Ryuhei and Ohta, Keisuke and Nakamura, Kei-Ichiro and Lang, Richard J
Journal: Pflügers Archiv-European Journal of Physiology (2017): 1--17
Authors: Hashitani, Hikaru and Nguyen, Michael J and Noda, Haruka and Mitsui, Retsu and Higashi, Ryuhei and Ohta, Keisuke and Nakamura, Kei-Ichiro and Lang, Richard J
Journal: Pflügers Archiv-European Journal of Physiology (2017): 1--17
Extensive Ca 2+ leak through K4750Q cardiac ryanodine receptors caused by cytosolic and luminal Ca 2+ hypersensitivity
Authors: Uehara, Akira and Murayama, Takashi and Yasukochi, Midori and Fill, Michael and Horie, Minoru and Okamoto, Toru and Matsuura, Yoshiharu and Uehara, Kiyoko and Fujimoto, Takahiro and Sakurai, Takashi and others, undefined
Journal: The Journal of general physiology (2017): 199--218
Authors: Uehara, Akira and Murayama, Takashi and Yasukochi, Midori and Fill, Michael and Horie, Minoru and Okamoto, Toru and Matsuura, Yoshiharu and Uehara, Kiyoko and Fujimoto, Takahiro and Sakurai, Takashi and others, undefined
Journal: The Journal of general physiology (2017): 199--218
Synchronicity and Rhythmicity of Purkinje Cell Firing during Generalized Spike-and-Wave Discharges in a Natural Mouse Model of Absence Epilepsy Complex Spike Synchronicity during GSWDs
Authors: Kros, Lieke and Lindeman, S and er , undefined and Eelkman Rooda, Oscar HJ and Murugesan, Pavithra and Bina, Lorenzo and Bosman, Laurens WJ and De Zeeuw, Chris I and Hoebeek, Freek E
Journal: Frontiers in Cellular Neuroscience (2017): 346
Authors: Kros, Lieke and Lindeman, S and er , undefined and Eelkman Rooda, Oscar HJ and Murugesan, Pavithra and Bina, Lorenzo and Bosman, Laurens WJ and De Zeeuw, Chris I and Hoebeek, Freek E
Journal: Frontiers in Cellular Neuroscience (2017): 346
Simultaneous Measurement of Neural Activities of Acute Mouse Hippocampal Slices Using Multi-Electrode Array System and Laser Confocal Calcium Imaging
Authors: Hamasaki, Yuuta and Haba, Natsumi and Iwata, Naoki and Uno, Yoshiki and Saito, Minoru
Journal: Journal of Behavioral and Brain Science (2017): 68
Authors: Hamasaki, Yuuta and Haba, Natsumi and Iwata, Naoki and Uno, Yoshiki and Saito, Minoru
Journal: Journal of Behavioral and Brain Science (2017): 68
Ca 2+ signals initiate at immobile IP 3 receptors adjacent to ER-plasma membrane junctions
Authors: Thillaiappan, Nagendra Babu and Chavda, Alap P and Tovey, Stephen C and Prole, David L and Taylor, Colin W
Journal: Nature Communications (2017): 1505
Authors: Thillaiappan, Nagendra Babu and Chavda, Alap P and Tovey, Stephen C and Prole, David L and Taylor, Colin W
Journal: Nature Communications (2017): 1505
Mechanical loading disrupts osteocyte plasma membranes which initiates mechanosensation events in bone
Authors: Yu, Kanglun and Sellman, David P and Bahraini, Anoosh and Hagan, Mackenzie L and Elsherbini, Ahmed and Vanpelt, Kayce T and Marshall, Peyton L and Hamrick, Mark W and McNeil, Anna and McNeil, Paul L and others, undefined
Journal: Journal of Orthopaedic Research (2017)
Authors: Yu, Kanglun and Sellman, David P and Bahraini, Anoosh and Hagan, Mackenzie L and Elsherbini, Ahmed and Vanpelt, Kayce T and Marshall, Peyton L and Hamrick, Mark W and McNeil, Anna and McNeil, Paul L and others, undefined
Journal: Journal of Orthopaedic Research (2017)
Neonatal CX26 removal impairs neocortical development and leads to elevated anxiety
Authors: Su, Xin and Chen, Jing-Jing and Liu, Lin-Yun and Huang, Qian and Zhang, Li-Zhao and Li, Xiao-Yang and He, Xiang-Nan and Lu, Wenlian and Sun, Shan and Li, Huawei and others, undefined
Journal: Proceedings of the National Academy of Sciences (2017): 201613237
Authors: Su, Xin and Chen, Jing-Jing and Liu, Lin-Yun and Huang, Qian and Zhang, Li-Zhao and Li, Xiao-Yang and He, Xiang-Nan and Lu, Wenlian and Sun, Shan and Li, Huawei and others, undefined
Journal: Proceedings of the National Academy of Sciences (2017): 201613237
Application notes
A Comparison of Fluorescent Red Calcium Indicators for Detecting Intracellular Calcium Mobilization in CHO Cells
A Meta-Analysis of Common Calcium Indicators
A New Red Fluorescent & Robust Screen Quest™ Rhod-4™ Ca2+Indicator for Screening GPCR & Ca2+ Channel Targets
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
A Meta-Analysis of Common Calcium Indicators
A New Red Fluorescent & Robust Screen Quest™ Rhod-4™ Ca2+Indicator for Screening GPCR & Ca2+ Channel Targets
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
A Novel NO Wash Probeniceid-Free Calcium Assay for Functional Analysis of GPCR and Calcium Channel Targets
FAQ
What are the differences between calcium ion indicators: Cal 520, Cal 520FF, and Cal 520N?
Are there any calcium indicators that don't require probenecid (PBC)?
Are there upgraded trypan blue derivatives for cell viability testing?
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?
Are there any calcium indicators that don't require probenecid (PBC)?
Are there upgraded trypan blue derivatives for cell viability testing?
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?