Calculator
Fura-2 AM
Fura-2 AM is a cell-permeable, ratiometric calcium ion indicator that emits green fluorescence upon UV excitation.
Fura-2 AM is the membrane permeable form of Fura-2. It belongs to a class of ratiometric calcium indicators, which also contain fluorescent dyes such as Indo-1. These probes quantitate calcium by observing changes at two wavelengths of a fluorophore upon calcium chelation and computing the ratio of these two points. This is in contrast to intensiometric probes, like Fluo-4, where a single spectral value is utilized.
Principles and mechanisms
Upon calcium binding, Fura-2 will exhibit an absorption shift in the 300 to 400 nm range, where the calcium free dye has an excitation maximum of 340 nm while the calcium bound dye has an excitation maximum of 380 nm. This change in absorbance can be measured by scanning the excitation spectrum, while monitoring the emission at 510 nm. The calculated 340/380 nm ratio is proportional to intracellular calcium ion concentrations.
Fura-2 vs Fluo-4
Ratiometric dyes like Fura-2 have several advantages over single-wavelength calcium probes, such as Fluo-4, namely, mitigating the effects of dye loading, leaking and photobleaching.
Fura-2 vs Indo-1
Fura-2 is an excitation ratiometric dye, wherein the absorbance spectra shifts (340/380 nm). Indo-1 is an emission ratiometric dye, wherein the emission spectra shifts (400/480 nm).
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
Prepare a 2 to 5 mM stock solution of Fura-2 AM in high-quality, anhydrous DMSO.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Fura-2 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Fura-2 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-2 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-2 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.
- Prepare cells in growth medium overnight.
On the next day, add 1X Fura-2 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.
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 = 340/510 nm cutoff 475 nm and Ex/Em2 = 380/510 nm cutoff 475 nm.
Calculators
Common stock solution preparation
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 99.814 µL | 499.072 µL | 998.143 µL | 4.991 mL | 9.981 mL |
| 5 mM | 19.963 µL | 99.814 µL | 199.629 µL | 998.143 µL | 1.996 mL |
| 10 mM | 9.981 µL | 49.907 µL | 99.814 µL | 499.072 µL | 998.143 µL |
Molarity calculator
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
| / | = | x | = |
Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) |
| Fura-8FF™, AM | 354 | 524 |
| Fura-FF, AM [Fura-2FF, AM] *CAS 348079-12-9* | 336 | 505 |
| Fura Red, AM *CAS 149732-62-7* | 435 | 639 |
| Fura-8™, AM | 354 | 524 |
| Rhod-2, AM *CAS#: 145037-81-6* | 553 | 577 |
| Rhod-2, AM *UltraPure Grade* *CAS#: 145037-81-6* | 553 | 577 |
| Fura-10™, AM | 354 | 524 |
Citations
Authors: Gan, Lu and Jiang, Qiwei and Huang, Dong and Wu, Xueji and Zhu, Xinying and Wang, Lei and Xie, Wei and Huang, Jialuo and Fan, Runzhu and Jing, Yihang and others,
Journal: Nature Chemical Biology (2024): 1--11
Authors: Triana, Miryam A Hortua and M{\'a}rquez-Nogueras, Karla M and Fazli, Mojtaba Sedigh and Quinn, Shannon and Moreno, Silvia NJ
Journal: Journal of Biological Chemistry (2024): 105771
Authors: Habib, Sofia
Journal: (2023)
Authors: Wang, Jie and Cui, Zong Jie
Journal: Biomedicines (2023): 885
Authors: Liu, Zhihui and Wang, Yu and Zhou, Chong and Xu, Qingyang and Gao, Hongxin and Huo, Mohan and Jiang, Xiaowen and Yu, Wenhui
Journal: (2022)
References
Authors: Shao M, Wang HM, Liu ZH, Shen P, Cai RX.
Journal: Wei Sheng Wu Xue Bao (2005): 805
Authors: Paredes-Gamero EJ, Franca JP, Moraes AA, Aguilar MO, Oshiro ME, Ferreira AT.
Journal: J Fluoresc (2004): 711
Authors: Xu T, Yang W, Huo XL, Song T.
Journal: J Biochem Biophys Methods (2004): 219
Authors: McConnell G, Riis E.
Journal: Phys Med Biol (2004): 4757
Authors: McHugh JM, Kenyon JL.
Journal: Am J Physiol Cell Physiol (2004): C342
Safety data sheet