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AAT Bioquest

Fura-10™, AM

Among ratiometric calcium ion indicators, Fura-2 and Indo-1 are the two most popular ones. However, there are still a few challenges for using these two calcium ion indicators, in particular, for live cells. UV-excitation of Fura 2 caused fast photobleaching. Fura-8™ was introduced a few years ago to shift the excitation closer to visible light. Although Fura-8 demonstrated significant improvement in the ratio of signal/background, it is not well retained in live cells just like Fura-2. Fura-10 have recently been introduced to address this cellular retention issue. Fura 10 demonstrated dramatic improvement in the ratio of signal/background in the absence of probenecid.

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-10™ AM stock solution
  1. Prepare a 2 to 5 mM Fura-10™ AM stock solution in high-quality, anhydrous DMSO.

PREPARATION OF WORKING SOLUTION

Fura-10™ AM working solution
  1. On the day of the experiment, either dissolve Fura-10™ AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

  2. Prepare a 2 to 20 µM Fura-10™ 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-10™ 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-10™ 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-10™ 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 containing a programmable liquid handling system such as a FlexStation, at Ex/Em1 = 354/524 nm cutoff 475 nm and Ex/Em2 = 415/524 nm cutoff 475 nm.

Spectrum

Citations

View all 2 citations: Citation Explorer
Single Cell Droplet-Based Efficacy and Transcriptomic Analysis of a Novel Anti-KLRG1 Antibody for Elimination of Autoreactive T Cells
Authors: Konry, Tania and Sulllivan, Matthew and Rozzo, Aldo and Ward, Antonio and Rao, Patricia and Soler-Ferran, Dulce and Greenberg, Steven
Journal: (2024)

References

View all 50 references: Citation Explorer
Histamine induces intracellular Ca2+ oscillations and nitric oxide release in endothelial cells from brain microvascular circulation.
Authors: Berra-Romani, Roberto and Faris, Pawan and Pellavio, Giorgia and Orgiu, Matteo and Negri, Sharon and Forcaia, Greta and Var-Gaz-Guadarrama, Verónica and Garcia-Carrasco, Mario and Botta, Laura and Sancini, Giulio and Laforenza, Umberto and Moccia, Francesco
Journal: Journal of cellular physiology (2020): 1515-1530
Pharmacological and genetic characterisation of the canine P2X4 receptor.
Authors: Sophocleous, Reece A and Berg, Tracey and Finol-Urdaneta, Rocio K and Sluyter, Vanessa and Keshiya, Shikara and Bell, Lachlan and Curtis, Stephen J and Curtis, Belinda L and Seavers, Aine and Bartlett, Rachael and Dowton, Mark and Stokes, Leanne and Ooi, Lezanne and Sluyter, Ronald
Journal: British journal of pharmacology (2020)
Acupotomy Alleviates Energy Crisis at Rat Myofascial Trigger Points.
Authors: Zhang, Yi and Du, Ning-Yu and Chen, Chen and Wang, Tong and Wang, Li-Juan and Shi, Xiao-Lu and Li, Shu-Ming and Guo, Chang-Qing
Journal: Evidence-based complementary and alternative medicine : eCAM (2020): 5129562
Reduced store-operated Ca2+ entry impairs mesenteric artery function in response to high external glucose in type 2 diabetic ZDF rats.
Authors: Schach, Christian and Wester, Michael and Leibl, Florian and Redel, Andreas and Gruber, Michael and Maier, Lars S and Endemann, Dierk and Wagner, Stefan
Journal: Clinical and experimental pharmacology & physiology (2020)
High-salt intake increases TRPC3 expression and enhances TRPC3-mediated calcium influx and systolic blood pressure in hypertensive patients.
Authors: Hu, Yingru and Xia, Weijie and Li, Yingsha and Wang, Qianran and Lin, Shaoyang and Wang, Bin and Zhou, Cui and Cui, Yuanting and Jiang, Yanli and Pu, Xiaona and Wei, Xiao and Wu, Hao and Zhang, Hengshu and Zhu, Zhiming and Liu, Daoyan and Li, Zhiyong
Journal: Hypertension research : official journal of the Japanese Society of Hypertension (2020)
Page updated on November 3, 2024

Ordering information

Price
Unit size
5x50 ug
1 mg
Catalog Number
Quantity
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Physical properties

Dissociation constant (Kd, nM)260

Molecular weight

1194.14

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
UNSPSC12171501

Platform

Fluorescence microplate reader

Excitation354 nm and 415 nm
Emission524 nm
Cutoff475 nm
Recommended plateBlack wall, Clear bottom
Instrument specification(s)Bottom read mode, Programmable liquid handling
<strong>ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells measured with </strong><strong>Fura-2 AM, Fura-8&trade; AM and Fura-10&trade; AM in the absence of Probenecid. </strong>CHO-K1cells were seeded overnight in 50,000 cells per 100 &micro;L per well in a 96-well black wall/clear bottom costar plate. 100 &micro;L of 5&nbsp;uM&nbsp;Fura-2 AM or Fura-8&trade; AM or Fura-10&trade; AM without probenecid was added into the cells, and the cells were incubated at 37 <sup>o</sup>C for 45 minutes and RT for 30 minutes. &nbsp;ATP (50&micro;L/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.
<strong>ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells measured with </strong><strong>Fura-2 AM, Fura-8&trade; AM and Fura-10&trade; AM in the absence of Probenecid. </strong>CHO-K1cells were seeded overnight in 50,000 cells per 100 &micro;L per well in a 96-well black wall/clear bottom costar plate. 100 &micro;L of 5&nbsp;uM&nbsp;Fura-2 AM or Fura-8&trade; AM or Fura-10&trade; AM without probenecid was added into the cells, and the cells were incubated at 37 <sup>o</sup>C for 45 minutes and RT for 30 minutes. &nbsp;ATP (50&micro;L/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.
<strong>ATP-stimulated calcium response of endogenous P2Y receptor in CHO-K1 cells measured with </strong><strong>Fura-2 AM, Fura-8&trade; AM and Fura-10&trade; AM in the absence of Probenecid. </strong>CHO-K1cells were seeded overnight in 50,000 cells per 100 &micro;L per well in a 96-well black wall/clear bottom costar plate. 100 &micro;L of 5&nbsp;uM&nbsp;Fura-2 AM or Fura-8&trade; AM or Fura-10&trade; AM without probenecid was added into the cells, and the cells were incubated at 37 <sup>o</sup>C for 45 minutes and RT for 30 minutes. &nbsp;ATP (50&micro;L/well) was added by FlexStation (Molecular Devices) to achieve the final indicated concentrations.