Calculator
Fura-8FF™, AM
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-8FF™ AM in high-quality, anhydrous DMSO.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Fura-8FF™ AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Fura-8FF™ 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-8FF™ 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-8FF™ 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-8FF™ 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 = 355/530 nm cutoff 475 nm and Ex/Em2 = 415/530 nm cutoff 475 nm.
Calculators
Common stock solution preparation
| 0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
| 1 mM | 102.684 µL | 513.421 µL | 1.027 mL | 5.134 mL | 10.268 mL |
| 5 mM | 20.537 µL | 102.684 µL | 205.368 µL | 1.027 mL | 2.054 mL |
| 10 mM | 10.268 µL | 51.342 µL | 102.684 µL | 513.421 µL | 1.027 mL |
Molarity calculator
| Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
Product family
| Name | Excitation (nm) | Emission (nm) |
| Fura-8™, AM | 354 | 524 |
| Fluo-8FF™, AM | 495 | 516 |
| Fura-10™, 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 Red, AM *CAS 149732-62-7* | 435 | 639 |
Citations
Authors: Li, Zhi Ying and Jiang, Wen Yi and Cui, Zong Jie
Journal: Photochemical & Photobiological Sciences (2015): 414--428
Authors: Liang, Hui Yuan and Song, Zhi Min and Cui, Zong Jie
Journal: Biochemical and biophysical research communications (2013): 361--367
References
Authors: Shao M, Wang HM, Liu ZH, Shen P, Cai RX.
Journal: Wei Sheng Wu Xue Bao (2005): 805
Authors: McHugh JM, Kenyon JL.
Journal: Am J Physiol Cell Physiol (2004): C342
Authors: Paredes-Gamero EJ, Franca JP, Moraes AA, Aguilar MO, Oshiro ME, Ferreira AT.
Journal: J Fluoresc (2004): 711
Authors: McConnell G, Riis E.
Journal: Phys Med Biol (2004): 4757
Authors: Xu T, Yang W, Huo XL, Song T.
Journal: J Biochem Biophys Methods (2004): 219
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