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AAT Bioquest

Fura-2, AM *CAS 108964-32-5*

Fluorescence excitation spectra of Fura-2 in solutions conctaining 0 to 39uM free Ca2+.
Fluorescence excitation spectra of Fura-2 in solutions conctaining 0 to 39uM free Ca2+.
Fluorescence excitation spectra of Fura-2 in solutions conctaining 0 to 39uM free Ca2+.
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Physical properties
Dissociation constant (Kd, nM)145
Molecular weight1001.86
SolventDMSO
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
UNSPSC12352200

OverviewpdfSDSpdfProtocol


CAS
108964-32-5
Molecular weight
1001.86
Dissociation constant (Kd, nM)
145
Excitation (nm)
336
Emission (nm)
505
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. Fura-2, AM is a cell-permeable calcium indicator that is emission-ratiometric and UV light-excitable. This AM ester form can be loaded into live cells noninvasively.

Platform


Fluorescence microscope

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

Fluorescence microplate reader

Excitation340, 380
Emission510
Cutoff475
Recommended plateBlack 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.

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

PREPARATION OF WORKING SOLUTION

Fura-2 AM Working Solution
On the day of the experiment, either dissolve Fura-2 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature. Prepare a dye working solution of 2 to 20 µM in a buffer of your choice (e.g., Hanks and Hepes buffer) with 0.04% Pluronic® F-127. For most cell lines, Fura-2 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-2 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 solution, 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 Fura-2 AM working solution into 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. 

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fura-2, AM *CAS 108964-32-5* 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 mM99.814 µL499.072 µL998.143 µL4.991 mL9.981 mL
5 mM19.963 µL99.814 µL199.629 µL998.143 µL1.996 mL
10 mM9.981 µL49.907 µL99.814 µL499.072 µL998.143 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


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spectrum

Spectral properties

Excitation (nm)336
Emission (nm)505

Images


Citations


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References


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
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
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