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AAT Bioquest

Cal-520FF™, AM

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.

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
  1. 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

Cal-520FF™ AM Working Solution
  1. 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.

  2. 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.

  1. Prepare cells in growth medium overnight.
  2. 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.

  3. 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.

  4. 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.
  5. 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 mg0.5 mg1 mg5 mg10 mg
1 mM87.802 µL439.012 µL878.025 µL4.39 mL8.78 mL
5 mM17.56 µL87.802 µL175.605 µL878.025 µL1.756 mL
10 mM8.78 µL43.901 µL87.802 µL439.012 µL878.025 µL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
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Spectrum

Product family

NameExcitation (nm)Emission (nm)Quantum yield
Cal-520®, AM4925150.751
Cal-520N™, AM4925150.751
Cal-520ER™ AM492515-
Cal-590™ AM5745880.621
Cal-630™ AM6096260.371
Cal-500™ AM3884820.481

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
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
Synergistic drug combination effectively blocks Ebola virus infection
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
High-Throughput Phenotyping of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Neurons Using Electric Field Stimulation and High-Speed Fluorescence Imaging
Authors: Daily, Neil J and Du, Zhong-Wei and Wakatsuki, Tetsuro
Journal: ASSAY and Drug Development Technologies (2017)
HTS-Compatible Voltage-and Ca 2+-Sensitive Dye Recordings from hiPSC-Derived Cardiomyocytes Using the Hamamatsu FDSS Systems
Authors: Kettenhofen, Ralf
Journal: Stem Cell-Derived Models in Toxicology (2017): 135--152
Page updated on October 12, 2024

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1 mg
10x50 ug
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Physical properties

Dissociation constant (Kd, nM)9800

Molecular weight

1138.92

Solvent

DMSO

Spectral properties

Excitation (nm)

492

Emission (nm)

515

Quantum yield

0.751

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

Fluorescence microscope

ExcitationFITC
EmissionFITC
Recommended plateBlack wall, clear bottom

Fluorescence microplate reader

Excitation490
Emission525
Cutoff515
Recommended plateBlack wall, clear bottom
Instrument specification(s)Bottom read mode, Programmable liquid handling