AAT Bioquest

Mag-Fluo-4 AM

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Physical properties
Dissociation constant (Kd, nM)22000
Molecular weight817.65
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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See also: Fluoresceins
Molecular weight
Dissociation constant (Kd, nM)
Extinction coefficient (cm -1 M -1)
Excitation (nm)
Emission (nm)
Quantum yield
The cell-permeant Mag-Fluo-4 AM is an analog of Fluo-4 AM with a Kd for Mg ion of 4.7 mM and a Kd for Ca ion of 22 µM, making it useful as an intracellular Mg ion indicator as well as a low-affinity Ca ion indicator. This low-affinity fluorescent Ca ion indicator has been used to accurately track the kinetics of the spatially averaged free Ca ion transient in skeletal muscle. Mag-fluo-4 yields reliable kinetic information about the spatially averaged free Ca ion transient in skeletal muscle.


Flow cytometer

Excitation488 nm laser
Emission530/30 nm filter
Instrument specification(s)FITC channel

Fluorescence microscope

ExcitationFITC filter set
EmissionFITC filter set
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

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


Mag-Fluo-4 AM Working Solution
  1. On the day of the experiment, either dissolve Mag-Fluo-4 AM in DMSO or thaw an aliquot of the indicator stock solution to room temperature.

  2. Prepare a 2 to 20 µM Mag-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, Mag-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 Mag-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 Mag-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 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 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 Ex/Em = 490/525 nm cutoff 515 nm.


Common stock solution preparation

Table 1. Volume of DMSO needed to reconstitute specific mass of Mag-Fluo-4 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 mM122.302 µL611.509 µL1.223 mL6.115 mL12.23 mL
5 mM24.46 µL122.302 µL244.603 µL1.223 mL2.446 mL
10 mM12.23 µL61.151 µL122.302 µL611.509 µL1.223 mL

Molarity calculator

Enter any two values (mass, volume, concentration) to calculate the third.

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Spectral properties

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



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