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Fura Red, AM *CAS 149732-62-7*

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.

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
  1. 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
  1. 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.

  2. Prepare a 2 to 20 µM Fura Red 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 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 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 Red 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 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 mg0.5 mg1 mg5 mg10 mg
1 mM91.828 µL459.141 µL918.282 µL4.591 mL9.183 mL
5 mM18.366 µL91.828 µL183.656 µL918.282 µL1.837 mL
10 mM9.183 µL45.914 µL91.828 µL459.141 µL918.282 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 8 citations: Citation Explorer
Ultrasound-mediated spatial and temporal control of engineered cells in vivo
Authors: Ivanovski, Filip and Me{\v{s}}ko, Maja and Lebar, Tina and Rupnik, Marko and Lain{\v{s}}{\v{c}}ek, Du{\v{s}}ko and Gradi{\v{s}}ek, Miha and Jerala, Roman and Ben{\v{c}}ina, Mojca
Journal: Nature Communications (2024): 7369
P-selectin-dependent leukocyte adhesion is governed by endolysosomal two-pore channel 2
Authors: Goretzko, Jonas and Pauels, Inga and Heitzig, Nicole and Thomas, Katharina and Kardell, Marina and Na{\ss}, Johannes and Krogsaeter, Einar Kleinhans and Schloer, Sebastian and Spix, Barbara and Matos, Anna L{\'\i}via Linard and others,
Journal: Cell Reports (2023): 113501
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

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
Page updated on November 9, 2024

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Unit size
1 mg
10x50 ug
Catalog Number
2104621048
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Physical properties

Dissociation constant (Kd, nM)400

Molecular weight

1088.99

Solvent

DMSO

Spectral properties

Excitation (nm)

435

Emission (nm)

639

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

CAS

149732-62-7

Platform

Fluorescence microplate reader

Excitation435, 470
Emission630, 650
CutoffEx, Em = 435, 630, cutoff 610. Ex, Em = 470, 650, cut off 630
Recommended plateBlack wall, clear bottom
Instrument specification(s)Bottom read mode, Programmable liquid handling
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