Fluo-4 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 Fluo-4 AM in high-quality, anhydrous DMSO.
PREPARATION OF WORKING SOLUTION
On the day of the experiment, either dissolve Fluo-4 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.
Prepare a 2 to 20 µM Fluo-4 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, Fluo-4 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 Fluo-4 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 Fluo-4 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 2 hours 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 FITC filter set or a fluorescence plate reader containing a programmable liquid handling system such as an FDSS, FLIPR, or FlexStation, at 490/525 nm cutoff 515 nm.
Calculators
Common stock solution preparation
0.1 mg | 0.5 mg | 1 mg | 5 mg | 10 mg | |
1 mM | 91.162 µL | 455.809 µL | 911.619 µL | 4.558 mL | 9.116 mL |
5 mM | 18.232 µL | 91.162 µL | 182.324 µL | 911.619 µL | 1.823 mL |
10 mM | 9.116 µL | 45.581 µL | 91.162 µL | 455.809 µL | 911.619 µL |
Molarity calculator
Mass (Calculate) | Molecular weight | Volume (Calculate) | Concentration (Calculate) | Moles | ||||
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Spectrum
Product family
Name | Excitation (nm) | Emission (nm) | Extinction coefficient (cm -1 M -1) | Quantum yield |
Fluo-3, AM *CAS 121714-22-5* | 506 | 515 | 86,0001 | 0.151 |
Fluo-3, AM *UltraPure grade* *CAS 121714-22-5* | 506 | 515 | 86,0001 | 0.151 |
Fluo-3, AM *Bulk package* *CAS 121714-22-5* | 506 | 515 | 86,0001 | 0.151 |
Fluo-3FF, AM *UltraPure grade* *Cell permeant* | 506 | 515 | 86,0001 | 0.151 |
Fluo-8H™, AM | 495 | 516 | 23430 | 0.161 |
Fluo-8L™, AM | 495 | 516 | 23430 | 0.161 |
Fluo-8FF™, AM | 495 | 516 | 23430 | 0.161 |
Fluo-5F, AM *Cell permeant* | 494 | 516 | - | - |
Fluo-5N, AM *Cell permeant* | 494 | 516 | - | - |
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Citations
Authors: Yuan, Tao and Wang, Yi and Wang, Haojue and Lu, Qizhen and Zhang, Xin and Li, Ziqing and Sun, Shui
Journal: Heliyon (2024)
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Journal: Bioscience Reports (2023)
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