AAT Bioquest

Fura-FF, AM [Fura-2FF, AM] *CAS 348079-12-9*

Fluorescence excitation spectra of Fura-2™ in the presence of 0 to 39 µM free Ca2+.
Fluorescence excitation spectra of Fura-2™ in the presence of 0 to 39 µM free Ca2+.
Fluorescence excitation spectra of Fura-2™ in the presence of 0 to 39 µM free Ca2+.
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Physical properties
Dissociation constant (Kd, nM)5500
Molecular weight1023.80
Spectral properties
Excitation (nm)336
Emission (nm)505
Storage, safety and handling
H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22
StorageFreeze (< -15 °C); Minimize light exposure


Molecular weight
Dissociation constant (Kd, nM)
Excitation (nm)
Emission (nm)
Among the ratiometric calcium indicators, Fura-2 and Indo-1 are most commonly used. Fura-2 is excitation-ratioable while Indo-1 is emission-ratioable. Fura-2 is preferred for ratio-imaging microscopy, in which it is more practical to change excitation wavelengths than emission wavelengths. Upon binding Ca2+, Fura-2 exhibits an absorption shift that can be observed by scanning the excitation spectrum between 300 and 400 nm, while monitoring the emission at ~510 nm. The cell-permeant Fura-2FF AM is an analog of Fura-2 AM with much lower calcium binding affinity, Kd ~10 µM. This AM ester form can be loaded into live cells noninvasively.


Fluorescence microscope

ExcitationFura 2 filter set
EmissionFura 2 filter set
Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Excitation340, 380
Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling

Example protocol


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

Fura-FF AM Stock Solution
  1. Prepare a 2 to 5 mM stock solution of Fura-FF AM in high-quality, anhydrous DMSO.


Fura-FF AM Working Solution
  1. On the day of the experiment, either dissolve Fura-FF AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

  2. Prepare a 2 to 20 µM Fura-FF 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, Fura-FF 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 Fura-FF 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.


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 Fura-FF 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 30 to 60 minutes.

    Note: Incubating the dye for longer than 1 hour 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 Fura 2 filter set or a fluorescence plate reader containing a programmable liquid handling system such as a FlexStation, at Ex/Em1 = 340/510 nm cutoff 475 nm and Ex/Em2 = 380/510 nm cutoff 475 nm.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fura-FF, AM [Fura-2FF, AM] *CAS 348079-12-9* 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 mM97.675 µL488.377 µL976.753 µL4.884 mL9.768 mL
5 mM19.535 µL97.675 µL195.351 µL976.753 µL1.954 mL
10 mM9.768 µL48.838 µL97.675 µL488.377 µL976.753 µL

Molarity calculator

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Spectral properties

Excitation (nm)336
Emission (nm)505



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View all 119 references: Citation Explorer
Load of calcium probe Fura -2/AM in Escherichia coli cells
Authors: Shao M, Wang HM, Liu ZH, Shen P, Cai RX.
Journal: Wei Sheng Wu Xue Bao (2005): 805
An Excel-based model of Ca2+ diffusion and fura 2 measurements in a spherical cell
Authors: McHugh JM, Kenyon JL.
Journal: Am J Physiol Cell Physiol (2004): C342
Problems caused by high concentration of ATP on activation of the P2X7 receptor in bone marrow cells loaded with the Ca2+ fluorophore fura-2
Authors: Paredes-Gamero EJ, Franca JP, Moraes AA, Aguilar MO, Oshiro ME, Ferreira AT.
Journal: J Fluoresc (2004): 711
Photonic crystal fibre enables short-wavelength two-photon laser scanning fluorescence microscopy with fura-2
Authors: McConnell G, Riis E.
Journal: Phys Med Biol (2004): 4757
Abnormal spectra alteration observed in Triton calibration method for measuring [Ca2+]i with fluorescence indicator, fura-2
Authors: Xu T, Yang W, Huo XL, Song T.
Journal: J Biochem Biophys Methods (2004): 219
Two-photon microscopy of fura-2-loaded cardiac myocytes with an all-solid-state tunable and visible femtosecond laser source
Authors: McConnell G, Smith GL, Girkin JM, Gurney AM, Ferguson AI.
Journal: Opt Lett (2003): 1742
AMPA-induced Ca(2+) influx in cultured rat cortical nonpyramidal neurones: pharmacological characterization using fura-2 microfluorimetry
Authors: Fischer W, Franke H, Scheibler P, Allgaier C, Illes P.
Journal: Eur J Pharmacol (2002): 53
Excitation wavelengths for fura 2 provide a linear relationship between [Ca(2+)] and fluorescence ratio
Authors: Palmer BM, Moore RL.
Journal: Am J Physiol Cell Physiol (2000): C1278
Tyrosine kinase inhibitors and Ca2+ signaling: direct interactions with fura-2
Authors: Berts A, Minneman KP.
Journal: Eur J Pharmacol (2000): 35
Selective measurement of endothelial or smooth muscle [Ca(2+)](i) in pressurized/perfused cerebral arteries with fura-2
Authors: Marrelli SP., undefined
Journal: J Neurosci Methods (2000): 145