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AAT Bioquest

Fura-8™, AM

Although Fura-2 has become the preferred excitation-ratioable calcium indicator of choice, it has certain limitations (e.g., lower sensitivity than single wavelength calcium indicators such as Fluo-8® and Cal-520®). To address these concerns, AAT Bioquest has devoted considerable efforts to the development of Fura™ 8, a high-affinity ratiometric calcium indicator with improved sensitivity and higher signal-to-noise ratios. The fluorescence emission of Fura™ 8 is red-shifted to a longer visible wavelength, facilitating the detection of Fura™ 8 by common filter sets. Fura™ 8, AM is membrane-permeant and is excited at 355 nm and 415 nm and emits at 530 nm.

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-8™ AM Stock Solution
  1. Prepare a 2 to 5 mM stock solution of Fura-8™ AM in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Fura-8™ AM Working Solution
  1. On the day of the experiment, either dissolve Fura-8™ AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

  2. Prepare a 2 to 20 µM Fura-8™ 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-8™ 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-8™ 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-8™ 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 = 355/530 nm cutoff 475 nm and Ex/Em2 = 415/530 nm cutoff 475 nm.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fura-8™, 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 mM105.053 µL525.265 µL1.051 mL5.253 mL10.505 mL
5 mM21.011 µL105.053 µL210.106 µL1.051 mL2.101 mL
10 mM10.505 µL52.527 µL105.053 µL525.265 µL1.051 mL

Molarity calculator

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

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum

Citations

View all 13 citations: Citation Explorer
TRPM8 overexpression suppresses hepatocellular carcinoma progression and improves survival by modulating the RTP3/STAT3 pathway
Authors: Chen, Lichan and Xu, Nansong and Gou, DongMei and Song, Jianning and Zhou, Mingqin and Zhang, Yajun and Zhang, Haohua and Zhu, Liwen and Huang, Weihong and Zhu, Yue and others,
Journal: Cancer Medicine (2024): e70109
HMGB1-Like Dorsal Switch Protein 1 Triggers a Damage Signal in Mosquito Gut to Activate Dual Oxidase via Eicosanoids
Authors: Ahmed, Shabbir and Sajjadian, Seyedeh Minoo and Kim, Yonggyun
Journal: Journal of Innate Immunity (2022): 1--16
Identification of G protein-coupled receptor 55 (GPR55) as a target of curcumin
Authors: Harada, Naoki and Okuyama, Mai and Teraoka, Yoshiaki and Arahori, Yumi and Shinmori, Yoh and Horiuchi, Hiroko and Luis, Paula B and Joseph, Akil I and Kitakaze, Tomoya and Matsumura, Shigenobu and others,
Journal: npj Science of Food (2022): 1--9
PGE2 mediates hemocyte-spreading behavior by activating aquaporin via cAMP and rearranging actin cytoskeleton via Ca2+
Authors: Ahmed, Shabbir and Kim, Yonggyun
Journal: Developmental \& Comparative Immunology (2021): 104230
TRPM4 links calcium signaling to membrane potential in pancreatic acinar cells
Authors: Diszh{\'a}zi, Gyula and Magyar, Zsuzsanna {\'E} and Lisztes, Erika and T{\'o}th-Moln{\'a}r, Edit and N{\'a}n{\'a}si, P{\'e}ter P and Vennekens, Rudi and T{\'o}th, Bal{\'a}zs I and Alm{\'a}ssy, J{\'a}nos
Journal: Journal of Biological Chemistry (2021)

References

View all 84 references: Citation Explorer
Measurement of [Ca2+] in cell suspensions using indo-1
Authors: Nelemans A., undefined
Journal: Methods Mol Biol (2006): 47
A flow cytometric comparison of Indo-1 to fluo-3 and Fura Red excited with low power lasers for detecting Ca(2+) flux
Authors: Bailey S, Macardle PJ.
Journal: J Immunol Methods (2006): 220
Ratiometric intracellular calcium imaging in the isolated beating rat heart using indo-1 fluorescence
Authors: Eerbeek O, Mik EG, Zuurbier CJ, van 't Loo M, Donkersloot C, Ince C.
Journal: J Appl Physiol (2004): 2042
Negative inotropic effects of angiotensin II, endothelin-1 and phenylephrine in indo-1 loaded adult mouse ventricular myocytes
Authors: Sakurai K, Norota I, Tanaka H, Kubota I, Tomoike H, Endo M.
Journal: Life Sci (2002): 1173
Usefulness of the analytic method of intracellular calcium and the problems--aequorin and indo-1 signal
Authors: Endoh M., undefined
Journal: Nippon Yakurigaku Zasshi (2000): 361
Page updated on November 9, 2024

Ordering information

Price
Unit size
1 mg
10x50 ug
Catalog Number
2105521056
Quantity
Add to cart

Additional ordering information

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Physical properties

Dissociation constant (Kd, nM)260

Molecular weight

951.90

Solvent

DMSO

Spectral properties

Excitation (nm)

354

Emission (nm)

524

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

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

Fluorescence microplate reader

Excitation355, 415
Emission530
Cutoff475
Recommended plateBlack wall, clear bottom
Instrument specification(s)Bottom read mode, Programmable liquid handling
ATP Dose response in CHO-K1 cells measured with Fura-2 AM and Fura-8&trade; AM respectively. CHO-K1 cells were seeded overnight at 40,000 cells/100 &micro;L/well in a black wall/clear bottom 96-well plate. The cells incubated with Fura-2 AM or Fura-8 AM calcium assay&nbsp; dye-loading solution respectively for 1 hour. ATP Dose was added by Flexstation.
ATP Dose response in CHO-K1 cells measured with Fura-2 AM and Fura-8&trade; AM respectively. CHO-K1 cells were seeded overnight at 40,000 cells/100 &micro;L/well in a black wall/clear bottom 96-well plate. The cells incubated with Fura-2 AM or Fura-8 AM calcium assay&nbsp; dye-loading solution respectively for 1 hour. ATP Dose was added by Flexstation.
ATP Dose response in CHO-K1 cells measured with Fura-2 AM and Fura-8&trade; AM respectively. CHO-K1 cells were seeded overnight at 40,000 cells/100 &micro;L/well in a black wall/clear bottom 96-well plate. The cells incubated with Fura-2 AM or Fura-8 AM calcium assay&nbsp; dye-loading solution respectively for 1 hour. ATP Dose was added by Flexstation.