Fura-8™, AM

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Physical properties

Dissociation constant (K_d, nM)	260
Molecular weight	951.90
Solvent	DMSO

Spectral properties

Excitation (nm)	354
Emission (nm)	524

Storage, safety and handling

H-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

Molecular weight

951.90

Dissociation constant (K_d, 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

Excitation	Fura 2 filter set
Emission	Fura 2 filter set
Recommended plate	Black wall/clear bottom

Fluorescence microplate reader

Excitation	355, 415
Emission	530
Cutoff	475
Recommended plate	Black 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

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

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. 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-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 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.

Prepare cells in growth medium overnight.
On the next day, add 1X Fura-8™ 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.
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.
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.
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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	105.053 µL	525.265 µL	1.051 mL	5.253 mL	10.505 mL
5 mM	21.011 µL	105.053 µL	210.106 µL	1.051 mL	2.101 mL
10 mM	10.505 µL	52.527 µL	105.053 µL	525.265 µL	1.051 mL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Excitation (nm)	354
Emission (nm)	524

Product Family

Name	Excitation (nm)	Emission (nm)
Fura-8FF™, AM	354	524
Fura-2, AM CAS 108964-32-5	336	505
Fura-2, AM UltraPure Grade CAS 108964-32-5	336	505
Fura-FF, AM [Fura-2FF, AM] CAS 348079-12-9	336	505
Fura Red, AM CAS 149732-62-7	435	639
Fluo-8®, AM	495	516
Fura-10™, AM	354	524

Images

Figure 1. 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.

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