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AAT Bioquest

Fura-8™, AM

ATP Dose response in CHO-K1 cells measured with Fura-2 AM and Fura-8™ AM respectively. CHO-K1 cells were seeded overnight at 40,000 cells/100 µL/well in a black wall/clear bottom 96-well plate. The cells incubated with Fura-2 AM or Fura-8 AM calcium assay  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™ AM respectively. CHO-K1 cells were seeded overnight at 40,000 cells/100 µL/well in a black wall/clear bottom 96-well plate. The cells incubated with Fura-2 AM or Fura-8 AM calcium assay  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™ AM respectively. CHO-K1 cells were seeded overnight at 40,000 cells/100 µL/well in a black wall/clear bottom 96-well plate. The cells incubated with Fura-2 AM or Fura-8 AM calcium assay  dye-loading solution respectively for 1 hour. ATP Dose was added by Flexstation.
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Physical properties
Dissociation constant (Kd, nM)260
Molecular weight951.90
SolventDMSO
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
StorageFreeze (< -15 °C); Minimize light exposure
UNSPSC12352200

OverviewpdfSDSpdfProtocol


Molecular weight
951.90
Dissociation constant (Kd, nM)
260
Excitation (nm)
354
Emission (nm)
524
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.

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

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
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Spectrum


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spectrum

Spectral properties

Excitation (nm)354
Emission (nm)524

Images


Citations


View all 12 citations: Citation Explorer
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
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
Eicosanoid-induced calcium signaling mediates cellular immune responses of Tenebrio molitor
Authors: Roy, Miltan Chandra and Kim, Yonggyun
Journal: Entomologia Experimentalis et Applicata (2021)
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)
Mechanisms regulating adult neurogenesis in the hypothalamus using a genetic inducible-approach to label and optogenetically stimulate hypothalamic neural progenitors
Authors: Peswani, Rahul Lekhraj
Journal: (2018)
Nifedipine stimulates proliferation and migration of different breast cancer cells by distinct pathways
Authors: Zhao, Tao and Guo, Dongqing and Gu, Yuchun and Ling, Yang
Journal: Molecular Medicine Reports (2017): 2259--2263
Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to the development of pulmonary arterial hypertension
Authors: Guo, Dongqing and Gu, Junzhong and Jiang, Hui and Ahmed, Asif and Zhang, Zhiren and Gu, Yuchun
Journal: Journal of molecular and cellular cardiology (2016): 179--187
Fiber optic biofluorometer for physiological research on muscle slices
Authors: Belz, Mathias and Dendorfer, Andreas and Werner, Jan and Lambertz, Daniel and Klein, Karl-Friedrich
Journal: (2016): 97020Q
Nifedipine promotes the proliferation and migration of breast cancer cells
Authors: Guo, Dong-Qing and Zhang, Hao and Tan, Sheng-Jiang and Gu, Yu-Chun
Journal: PloS one (2014): e113649

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
Comment on "Usefulness of intracellular calcium analysis and the problem--aequorin and indo-1 signal"
Authors: Imaizumi Y., undefined
Journal: Nippon Yakurigaku Zasshi (2000): 101
Concentrations of caffeine greater than 20 mM increase the indo-1 fluorescence ratio in a Ca(2+)-independent manner
Authors: McKemy DD, Welch W, Airey JA, Sutko JL.
Journal: Cell Calcium (2000): 117
Intracellular calcium signals measured with indo-1 in isolated skeletal muscle fibres from control and mdx mice
Authors: Collet C, Allard B, Tourneur Y, Jacquemond V.
Journal: J Physiol (1999): 417
Measurement of [Ca2+]i in cell suspensions using indo-1
Authors: Nelemans A., undefined
Journal: Methods Mol Biol (1999): 41
Alpha-stat calibration of indo-1 fluorescence and measurement of intracellular free calcium in rat ventricular cells at different temperatures
Authors: Wang SQ, Zhou ZQ.
Journal: Life Sci (1999): 871