AAT Bioquest

Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3*

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Physical properties
Dissociation constant (Kd, nM)345
Molecular weight1096.95
Spectral properties
Extinction coefficient (cm -1 M -1)82000
Excitation (nm)495
Emission (nm)528
Quantum yield0.161
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
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Molecular weight
Dissociation constant (Kd, nM)
Extinction coefficient (cm -1 M -1)
Excitation (nm)
Emission (nm)
Quantum yield
Calcium measurement is critical for numerous biological investigations. Fluorescent probes that show spectral responses upon binding calcium have enabled researchers to investigate changes in intracellular free calcium concentrations by using fluorescence microscopy, flow cytometry, fluorescence spectroscopy and fluorescence microplate readers. Fluo-3 and Fluo-4 are most commonly used among the visible light-excitable calcium indicators. Fluo-4 is an analog of Fluo-3 with the two chlorine substituents replaced by fluorines, which results in increased fluorescence excitation at 488 nm and consequently higher fluorescence signal levels. Cells may be loaded with the AM ester forms of these calcium indicators by adding the dissolved indicator directly to dishes containing cultured cells. However, Fluo-3 AM and Fluo-4 AM are only moderately fluorescent in live cells upon esterase hydrolysis, and require harsh cell loading conditions to maximize their cellular calcium responses. Fluo-8® and Cal-520® calcium dyes have been developed to improve cell loading and calcium response while maintaining the convenient Fluo-3 and Fluo-4 spectral wavelength of maximum excitation @ ~490 nm and maximum emission @ ~520 nm.


Fluorescence microscope

Recommended plateBlack wall/clear bottom

Fluorescence microplate reader

Recommended plateBlack wall/clear bottom
Instrument specification(s)Bottom read mode/Programmable liquid handling

Example protocol


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

Fluo-4 AM *UltraPure grade* Stock Solution
  1. Prepare a 2 to 5 mM stock solution of Fluo-4 AM in high-quality, anhydrous DMSO.


Fluo-4 AM *UltraPure grade* Stock Solution
  1. 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.

  2. 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.


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 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.

  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 2 hours 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 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.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Fluo-4 AM *Ultrapure Grade* *CAS 273221-67-3* 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 mM91.162 µL455.809 µL911.619 µL4.558 mL9.116 mL
5 mM18.232 µL91.162 µL182.324 µL911.619 µL1.823 mL
10 mM9.116 µL45.581 µL91.162 µL455.809 µL911.619 µL

Molarity calculator

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Spectral properties

Extinction coefficient (cm -1 M -1)82000
Excitation (nm)495
Emission (nm)528
Quantum yield0.161

Product Family

NameExcitation (nm)Emission (nm)Extinction coefficient (cm -1 M -1)Quantum yield
Fluo-3, AM *CAS 121714-22-5*50651586,00010.151
Fluo-3, AM *UltraPure grade* *CAS 121714-22-5*50651586,00010.151
Fluo-3, AM *Bulk package* *CAS 121714-22-5*50651586,00010.151
Fluo-3FF, AM *UltraPure grade* *Cell permeant*50651586,00010.151
Fluo-8H™, AM495516234300.161
Fluo-8L™, AM495516234300.161
Fluo-8FF™, AM495516234300.161
Fluo-5F, AM *Cell permeant*494516--
Fluo-5N, AM *Cell permeant*494516--
Rhod-4™, AM523551-0.11
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