Cal-520®, AM
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-520® AM in high-quality, anhydrous DMSO.
Note: When reconstituted in DMSO, Cal-520® AM is a clear, colorless solution.
PREPARATION OF WORKING SOLUTION
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.
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.
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-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.
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 | 90.666 µL | 453.33 µL | 906.659 µL | 4.533 mL | 9.067 mL |
5 mM | 18.133 µL | 90.666 µL | 181.332 µL | 906.659 µL | 1.813 mL |
10 mM | 9.067 µL | 45.333 µL | 90.666 µL | 453.33 µL | 906.659 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
Product family
Name | Excitation (nm) | Emission (nm) | Quantum yield |
Cal-520® alkyne | 492 | 515 | 0.751 |
Cal-520® amine | 492 | 515 | 0.751 |
Cal-520® azide | 492 | 515 | 0.751 |
Cal-520® maleimide | 492 | 515 | 0.751 |
Cal-520ER™ AM | 492 | 515 | - |
Cal-520FF™, AM | 492 | 515 | 0.751 |
Cal-520N™, AM | 492 | 515 | 0.751 |
Cal-500™ AM | 388 | 482 | 0.481 |
Cal-590™ AM | 574 | 588 | 0.621 |
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Citations
Authors: TRAN VAN NHIEU, Guy and GUO, Fangrui and GUEVARA, Roberto and OUSSAEIDINE, Linda and DUPONT, Genevi{\`e}ve and COMBETTES, Laurent
Journal: bioRxiv (2025): 2025--08
Authors: Mistry, Adam and Pravica, Nika B and Avolio, Elisa and Mastitskaya, Svetlana and James, Andrew F
Journal: (2025)
Authors: Li, Junjun and Mu, Yongli and Chen, Yinqi and Zhang, Xuanhao and Wang, Yechun and Wang, Jiafeng and Ying, Jiajia and Yang, Hang and Zhou, Xuefei and Du, Yushen and others,
Journal: ACS nano (2025)
Authors: Kong, Yvonne X and Rehan, Rajan and Moreno, Cesar L and Madsen, S{\o}ren and Zhang, Yunwei and Zhao, Huiwen and Qi, Miao and Houlahan, Callum B and Cartland, Si{\^a}n P and Robertshaw, Declan and others,
Journal: The Journal of clinical investigation (2025)
Authors: Larsson, Pia and McGovern, Abigail and Tarlac, Volga and Setiabakti, Natasha M and Parker, Louis and Cody, Stephen H and Nunez-Iglesias, Juan and Fax{\"a}lv, Lars and Wittberg, Lisa Prahl and Hamilton, Justin R and others,
Journal: Journal of Thrombosis and Haemostasis (2025)
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