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Fura-10™, AM

<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.
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Physical properties
Dissociation constant (Kd, nM)260
Molecular weight1194.14
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
UNSPSC12171501

OverviewpdfSDSpdfProtocol


Molecular weight
1194.14
Dissociation constant (Kd, nM)
260
Excitation (nm)
354
Emission (nm)
524
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.

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

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.

Calculators


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fura-10™, 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 mM83.742 µL418.711 µL837.423 µL4.187 mL8.374 mL
5 mM16.748 µL83.742 µL167.485 µL837.423 µL1.675 mL
10 mM8.374 µL41.871 µL83.742 µL418.711 µL837.423 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


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spectrum

Spectral properties

Excitation (nm)354
Emission (nm)524

Images


References


View all 50 references: Citation Explorer
Rumex acetosa modulates platelet function and inhibits thrombus formation in rats.
Authors: Jeong, Dahye and Irfan, Muhammad and Lee, Dong-Ha and Hong, Seung-Bok and Oh, Jae-Wook and Rhee, Man Hee
Journal: BMC complementary medicine and therapies (2020): 98
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)
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)
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
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)
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
Low concentration of morphine protects against cell death, oxidative stress and calcium accumulation by nicotine in PC12 cells.
Authors: Amini, K and Zhaleh, H and Tahvilian, R and Farnia, V
Journal: Bratislavske lekarske listy (2019): 256-262
NHE8 attenuates Ca2+ influx into NRK cells and the proximal tubule epithelium.
Authors: Wiebe, Shane A and Plain, Allein and Pan, Wanling and O'Neill, Debbie and Braam, Branko and Alexander, R Todd
Journal: American journal of physiology. Renal physiology (2019): F240-F253
A3 receptor agonist, Cl-IBMECA, potentiate glucose-induced insulin secretion from MIN6 insulinoma cells possibly through transient Ca2+ entry.
Authors: Keyvanloo Shahrestanaki, Mohammad and Aghaei, Mahmoud
Journal: Research in pharmaceutical sciences (2019): 107-114
TRPV4 plays an important role in rat prefrontal cortex changes induced by acute hypoxic exercise.
Authors: Huang, Xing and Hu, Yanxin and Zhao, Li and Gu, Boya and Zhu, Rongxin and Li, Yan and Yang, Yun and Han, Tianyu and Yu, Jiabei and Mu, Lianwei and Han, Peng and Li, Cui and Zhang, Weijia and Hu, Yang
Journal: Saudi journal of biological sciences (2019): 1194-1206