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Mag-Fluo-4 AM

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.

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

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.

PREPARATION OF WORKING SOLUTION

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.

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.

  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.

Calculators

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.

Mass (Calculate)Molecular weightVolume (Calculate)Concentration (Calculate)Moles
/=x=

Spectrum

Citations

View all 15 citations: Citation Explorer
TRPV1 alleviates APOE4-dependent microglial antigen presentation and T cell infiltration in Alzheimer's disease
Authors: Lu, Jia and Wu, Kexin and Sha, Xudong and Lin, Jiayuan and Chen, Hongzhuan and Yu, Zhihua
Journal: Translational Neurodegeneration (2024): 52
African Swine Fever Virus Modulates the Endoplasmic Reticulum Stress-ATF6-Calcium Axis to Facilitate Viral Replication: ASFV modulates ER stress-ATF6-Ca2+ axis for replication
Authors: Wang, Yanjin and Li, Jiaqi and Cao, Hongwei and Li, Lian-Feng and Dai, Jingwen and Cao, Mengxiang and Deng, Hao and Zhong, Dailang and Luo, Yuzi and Li, Yongfeng and others,
Journal: Emerging Microbes \& Infections (2024): 2399945
Sulfated Laminarin Polysaccharides Reduce the Adhesion of Nano-COM Crystals to Renal Epithelial Cells by Inhibiting Oxidative and Endoplasmic Reticulum Stress
Authors: He, Tian-Qu and Wang, Zhi and Li, Chuang-Ye and Zhao, Yao-Wang and Tong, Xin-Yi and Liu, Jing-Hong and Ouyang, Jian-Ming
Journal: Pharmaceuticals (2024): 805
Mitochondrial-dependent oxidative phosphorylation is key for postnatal metabolic adaptation of alveolar macrophages in the lung
Authors: Zhang, Jun and Peng, Yu and Song, Haosen and Liu, Siqi and Li, Chuanwei and Zhang, Yi and Shi, Xiaowei and Guo, Huifang and Xu, Yingping
Journal: International Immunopharmacology (2024): 112012
NNAT is a novel mediator of oxidative stress that suppresses ER+ breast cancer
Authors: Plasterer, Cody and Semenikhina, Marharyta and Tsaih, Shirng-Wern and Flister, Michael J and Palygin, Oleg
Journal: Molecular Medicine (2023): 1--12

References

View all 25 references: Citation Explorer
Real-time intra-store confocal Ca2+ imaging in isolated mouse cardiomyocytes
Authors: Fern, undefined and ez-Tenorio M, Niggli E.
Journal: Cell Calcium. (2016)
Redistribution of subcellular calcium and its effect on apoptosis in primary cultures of rat proximal tubular cells exposed to lead
Authors: Wang H, Wang ZK, Jiao P, Zhou XP, Yang DB, Wang ZY, Wang L.
Journal: Toxicology (2015): 137
Role of Mitofusin-2 in mitochondrial localization and calcium uptake in skeletal muscle
Authors: Ainbinder A, Boncompagni S, Protasi F, Dirksen RT.
Journal: Cell Calcium (2015): 14
Tetanic Ca2+ transient differences between slow- and fast-twitch mouse skeletal muscle fibres: a comprehensive experimental approach
Authors: Calderon JC, Bolanos P, Caputo C.
Journal: J Muscle Res Cell Motil (2014): 279
TRPC1 is involved in Ca(2)(+) influx and cytotoxicity following Pb(2)(+) exposure in human embryonic kidney cells
Authors: Zhang H, Li W, Xue Y, Zou F.
Journal: Toxicol Lett (2014): 52
Page updated on December 11, 2024

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Physical properties

Dissociation constant (Kd, nM)22000

Molecular weight

817.65

Solvent

DMSO

Spectral properties

Extinction coefficient (cm -1 M -1)

82000

Excitation (nm)

495

Emission (nm)

528

Quantum yield

0.161

Storage, safety and handling

H-phraseH303, H313, H333
Hazard symbolXN
Intended useResearch Use Only (RUO)
R-phraseR20, R21, R22

Storage

Freeze (< -15 °C); Minimize light exposure
UNSPSC12352200

Platform

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

Excitation490
Emission525
Cutoff515
Recommended plateBlack wall, clear bottom
Instrument specification(s)Bottom read mode, Programmable liquid handling