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Fura-2 AM

UltraPure Grade; CAS 108964-32-5
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.

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

PREPARATION OF WORKING SOLUTION

Fura-2 AM *UltraPure Grade* Working Solution
  1. 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.

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

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fura-2, AM *UltraPure Grade* *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

Citations

View all 18 citations: Citation Explorer
A natural small molecule alleviates liver fibrosis by targeting apolipoprotein L2
Authors: Gan, Lu and Jiang, Qiwei and Huang, Dong and Wu, Xueji and Zhu, Xinying and Wang, Lei and Xie, Wei and Huang, Jialuo and Fan, Runzhu and Jing, Yihang and others,
Journal: Nature Chemical Biology (2024): 1--11
Calcium signals in the murine zona glomerulosa
Authors: Dinh, Hoang An
Journal: (2024)
Regulation of Calcium entry by cyclic GMP signaling in Toxoplasma gondii
Authors: Triana, Miryam A Hortua and M{\'a}rquez-Nogueras, Karla M and Fazli, Mojtaba Sedigh and Quinn, Shannon and Moreno, Silvia NJ
Journal: Journal of Biological Chemistry (2024): 105771
BK channels sustain neuronal Ca2+ oscillations to support hippocampal long-term potentiation and memory formation
Authors: Pham, Thomas and Hussein, Tamara and Calis, Dila and Bischof, Helmut and Skrabak, David and Cruz Santos, Melanie and Maier, Selina and Sp{\"a}hn, David and Kalina, Daniel and Simonsig, Stefanie and others,
Journal: Cellular and Molecular Life Sciences (2023): 369

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
Page updated on October 10, 2024

Ordering information

Price
AvailabilityIn stock
GradeUltraPure
Pure
UltraPure
Unit size
1 mg
20x50 ug
Catalog Number
Quantity
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Physical properties

Dissociation constant (Kd, nM)145

Molecular weight

1001.86

Solvent

DMSO

Spectral properties

Excitation (nm)

336

Emission (nm)

505

Storage, safety and handling

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

Storage

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

CAS

108964-32-5

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
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+.

Alternative formats