Cal-520FF™, AM
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Telephone | 1-800-990-8053 |
Fax | 1-800-609-2943 |
sales@aatbio.com | |
International | See distributors |
Bulk request | Inquire |
Custom size | Inquire |
Shipping | Standard overnight for United States, inquire for international |
Dissociation constant (Kd, nM) | 9800 |
Molecular weight | 1138.92 |
Solvent | DMSO |
Excitation (nm) | 493 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
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 |
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
Prepare a 2 to 5 mM stock solution of Cal-520FF™ AM in high-quality, anhydrous DMSO.
Note: When reconstituted in DMSO, Cal-520FF™ AM is a clear, colorless solution.
PREPARATION OF 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 2 to 20 µM Cal-520FF™ 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-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 solutions, 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 to 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
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
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum

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
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: Bl{\"o}mer, Laila Ananda and Filipis, Luiza and Canepari, Marco
Journal: Journal of Fluorescence (2021): 619--623
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: 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: 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: 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: Hamasaki, Yuuta and Haba, Natsumi and Iwata, Naoki and Uno, Yoshiki and Saito, Minoru
Journal: Journal of Behavioral and Brain Science (2017): 68
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: 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: 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
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