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) | 492 |
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) 492 | 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
![spectrum](/_next/image?url=https%3A%2F%2Fimages.aatbio.com%2Fspectra%2Fcal_520.png&w=2048&q=50)
Spectral properties
Excitation (nm) | 492 |
Emission (nm) | 515 |
Quantum yield | 0.751 |
Product Family
Name | Excitation (nm) | Emission (nm) | Quantum yield |
Cal-520®, AM | 492 | 515 | 0.751 |
Cal-520N™, AM | 492 | 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: Sun, Wei and He, Shihua and Martínez-Romero, Carles and Kouznetsova, Jennifer and Tawa, Gregory and Xu, Miao and Shinn, Paul and Fisher, Ethan G and Long, Yan and Motabar, Omid and others, undefined
Journal: Antiviral Research (2017): 165--172
Authors: Daily, Neil J and Du, Zhong-Wei and Wakatsuki, Tetsuro
Journal: ASSAY and Drug Development Technologies (2017)
Authors: Kettenhofen, Ralf
Journal: Stem Cell-Derived Models in Toxicology (2017): 135--152
Authors: Servin-Vences, M Rocio and Moroni, Mirko and Lewin, Gary R and Poole, Kate
Journal: eLife (2017): e21074
Authors: Mitsui, Retsu and Hashitani, Hikaru
Journal: Pflügers Archiv-European Journal of Physiology (2017): 1--14
Authors: Pan, Libiao and Yang, Junhua and Yang, Qian and Wang, Xiaomeng and Zhu, Liya and Liu, Yali and Lou, Huifang and Xu, Chou and Shen, Ying and Wang, Hao
Journal: Frontiers in molecular neuroscience (2017)
Authors: Shi, Xuefeng and Barchini, Jad and Ledesma, Hector Acaron and Koren, David and Jin, Yanjiao and Liu, Xiaorong and Wei, Wei and Cang, Jianhua
Journal: Nature Neuroscience (2017)
Authors: Kodama, Daisuke and Hirai, Takao and Kondo, Hisataka and Hamamura, Kazunori and Togari, Akifumi
Journal: FEBS Letters (2017)
Application notes
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