Fura Red, AM CAS 149732-62-7

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Physical properties

Dissociation constant (K_d, nM)	400
Molecular weight	1088.99
Solvent	DMSO

Spectral properties

Excitation (nm)	435
Emission (nm)	639

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

CAS

149732-62-7

Molecular weight

1088.99

Dissociation constant (K_d, nM)

400

Excitation (nm)

435

Emission (nm)

639

Fura Red is a visible light-excitable fura-2 analog that offers unique possibilities for ratiometric measurement of calcium ion in single cells by microphotometry, imaging or flow cytometry when used with single excitation, green-fluorescent calcium indicators. Fura Red AM is the cell-permeable version of Fura Red used for noninvasive intracellular loading. Fura Red AM can be simultaneously loaded into cells with Fluo-3 AM, Fluo-8 AM or Cal-520 AM. An advantage of combining two calcium dyes is that dyes with longer excitation wavelengths can be used. This usually causes less harm to the cells than using ratiometric dyes that are excited with UV- or near UV-light (e.g. Fura-2), as light at visible wavelengths is less phototoxic.

Platform

Fluorescence microplate reader

Excitation	435, 470
Emission	630, 650
Cutoff	Ex/Em = 435/630, cutoff 610. Ex/Em = 470/650, cut off 630
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 Red AM Stock Solution

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

PREPARATION OF WORKING SOLUTION

Fura Red AM Working Solution

On the day of the experiment, either dissolve Fura Red 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 Red 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 Red 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 Red 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 monitor fluorescence intensity using a fluorescence plate reader, which contains a programmable liquid handling system such as a FlexStation, at Ex/Em₁ = 435/630 nm cutoff 610 nm and Ex/Em₂ = 470/650 nm cutoff 630 nm.

Calculators

Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fura Red, AM *CAS 149732-62-7* 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	91.828 µL	459.141 µL	918.282 µL	4.591 mL	9.183 mL
5 mM	18.366 µL	91.828 µL	183.656 µL	918.282 µL	1.837 mL
10 mM	9.183 µL	45.914 µL	91.828 µL	459.141 µL	918.282 µL

Molarity calculator

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

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

Spectrum

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Spectral properties

Excitation (nm)	435
Emission (nm)	639

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-8™, AM	354	524
Calcein Red™ AM	562	576
Fura-10™, AM	354	524

Images

Figure 1. Chemical structure for Fura Red, AM *CAS 149732-62-7*

Citations

View all 5 citations: Citation Explorer

Acute and long-term effects of cannabinoids on hypertension and kidney injury
Authors: Golosova, Daria and Levchenko, Vladislav and Kravtsova, Olha and Palygin, Oleg and Staruschenko, Alexander
Journal: Scientific reports (2022): 1--12

Arachidonic acid-regulated calcium signaling in T cells from patients with rheumatoid arthritis promotes synovial inflammation
Authors: Ye, Zhongde and Shen, Yi and Jin, Ke and Qiu, Jingtao and Hu, Bin and Jadhav, Rohit R and Sheth, Khushboo and Weyand, Cornelia M and Goronzy, J{\"o}rg J
Journal: Nature communications (2021): 1--17

Cellular and Subcellular Mechanisms of Ventricular Mechano-Arrhythmogenicity
Authors: Cameron, Breanne Ashleigh
Journal: (2021)

Role of opioid signaling in kidney damage during the development of salt-induced hypertension
Authors: Golosova, Daria and Palygin, Oleg and Bohovyk, Ruslan and Klemens, Christine A and Levchenko, Vladislav and Spires, Denisha R and Isaeva, Elena and El-Meanawy, Ashraf and Staruschenko, Alexander
Journal: Life science alliance (2020)

TRPA1 channels are a source of calcium-driven cardiac mechano-arrhythmogenicity
Authors: Cameron, Breanne A and Stoyek, Matthew R and Bak, Jessi J and Quinn, T Alexander
Journal: bioRxiv (2020)

References

View all 12 references: Citation Explorer

Ratiometric analysis of fura red by flow cytometry: a technique for monitoring intracellular calcium flux in primary cell subsets
Authors: Wendt ER, Ferry H, Greaves DR, Keshav S.
Journal: PLoS One (2015): e0119532

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

Use of co-loaded Fluo-3 and Fura Red fluorescent indicators for studying the cytosolic Ca(2+)concentrations distribution in living plant tissue
Authors: Walczysko P, Wagner E, Albrechtova JT.
Journal: Cell Calcium (2000): 23

Monitoring calcium in outer hair cells with confocal microscopy and fluorescence ratios of fluo-3 and fura-red
Authors: Su ZL, Li N, Sun YR, Yang J, Wang IM, Jiang SC.
Journal: Shi Yan Sheng Wu Xue Bao (1998): 323

Problems associated with using Fura-2 to measure free intracellular calcium concentrations in human red blood cells
Authors: Blackwood AM, Sagnella GA, Mark and u ND, MacGregor GA.
Journal: J Hum Hypertens (1997): 601

Calcium transient alternans in blood-perfused ischemic hearts: observations with fluorescent indicator fura red
Authors: Wu Y, Clusin WT.
Journal: Am J Physiol (1997): H2161

IgG-induced Ca2+ oscillations in differentiated U937 cells; a study using laser scanning confocal microscopy and co-loaded fluo-3 and fura-red fluorescent probes
Authors: Floto RA, Mahaut-Smith MP, Somasundaram B, Allen JM.
Journal: Cell Calcium (1995): 377

Localization of calcium entry through calcium channels in olfactory receptor neurones using a laser scanning microscope and the calcium indicator dyes Fluo-3 and Fura-Red
Authors: Schild D, Jung A, Schultens HA.
Journal: Cell Calcium (1994): 341

Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo-3/Fura Red fluorescence ratios
Authors: Novak EJ, Rabinovitch PS.
Journal: Cytometry (1994): 135

The distribution of intracellular calcium chelator (fura-2) in a population of intact human red cells
Authors: Lew VL, Etzion Z, Bookchin RM, daCosta R, Vaananen H, Sassaroli M, Eisinger J.
Journal: Biochim Biophys Acta (1993): 152