Mag-Fluo-4 AM

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Additional ordering information

Telephone	1-800-990-8053
Fax	1-800-609-2943
Email	sales@aatbio.com
Quotation	Request
International	See distributors
Shipping	Standard overnight for United States, inquire for international

Physical properties

Dissociation constant (K_d, 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-phrase	H303, H313, H333
Hazard symbol	XN
Intended use	Research Use Only (RUO)
R-phrase	R20, R21, R22
Storage	Freeze (< -15 °C); Minimize light exposure
UNSPSC	12352200

Overview SDS Protocol

Molecular weight

817.65

Dissociation constant (K_d, nM)

22000

Extinction coefficient (cm ^-1 M ^-1)

82000

Excitation (nm)

495

Emission (nm)

528

Quantum yield

0.16¹

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.

Platform

Flow cytometer

Excitation	488 nm laser
Emission	530/30 nm filter
Instrument specification(s)	FITC channel

Fluorescence microscope

Excitation	FITC filter set
Emission	FITC filter set
Recommended plate	Black wall/clear bottom

Fluorescence microplate reader

Excitation	490
Emission	525
Cutoff	515
Recommended plate	Black wall/clear bottom
Instrument specification(s)	Bottom read mode/Programmable liquid handling

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

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

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. Prepare a dye working solution of 2 to 20 µM 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 to 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 solution, 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 Mag-Fluo-4 AM working solution into 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 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 mg	0.5 mg	1 mg	5 mg	10 mg
1 mM	122.302 µL	611.509 µL	1.223 mL	6.115 mL	12.23 mL
5 mM	24.46 µL	122.302 µL	244.603 µL	1.223 mL	2.446 mL
10 mM	12.23 µL	61.151 µL	122.302 µL	611.509 µL	1.223 mL

Molarity calculator

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

Mass (Calculate)		Molecular weight		Volume (Calculate)		Concentration (Calculate)		Moles
	/		=		x		=

Spectrum

Open in Advanced Spectrum Viewer

Spectral properties

Extinction coefficient (cm ^-1 M ^-1)	82000
Excitation (nm)	495
Emission (nm)	528
Quantum yield	0.16¹

Images

Figure 1. Chemical structure for Mag-Fluo-4 AM

Citations

View all 8 citations: Citation Explorer

The interaction between Hsp90-mediated unfolded protein response and autophagy contributes to As3+/Se4+ combination-induced apoptosis of acute promyelocytic leukemia cells
Authors: Chang, Jiayin and Yan, Shihai and Geng, Zhirong and Wang, Zhilin
Journal: Toxicology and Applied Pharmacology (2023): 116511

Reticulon 2 promotes gastric cancer metastasis via activating endoplasmic reticulum Ca2+ efflux-mediated ERK signalling
Authors: Song, Shushu and Liu, Bo and Zeng, Xiaoqing and Wu, Yingying and Chen, Hao and Wu, Hao and Gu, Jianxin and Gao, Xiaodong and Ruan, Yuanyuan and Wang, Hongshan
Journal: Cell death \& disease (2022): 1--13

Mitochondrial Calcium-Triggered Oxidative Stress and Developmental Defects in Dopaminergic Neurons Differentiated from Deciduous Teeth-Derived Dental Pulp Stem Cells with MFF Insufficiency
Authors: Sun, Xiao and Dong, Shuangshan and Kato, Hiroki and Kong, Jun and Ito, Yosuke and Hirofuji, Yuta and Sato, Hiroshi and Kato, Takahiro A and Sakai, Yasunari and Ohga, Shouichi and others,
Journal: Antioxidants (2022): 1361

SYNJ2BP Improves the Production of Lentiviral Envelope Protein by Facilitating the Formation of Mitochondrion-Associated Endoplasmic Reticulum Membrane
Authors: Duan, Yingyi and Wang, Xinhui and Sun, Kehui and Lin, Yuezhi and Wang, Xuefeng and Chen, Kewei and Yang, Guangpu and Wang, Xiaojun and Du, Cheng
Journal: Journal of Virology (2022): e00549--22

The three-spot seahorse-derived peptide PAGPRGPA attenuates ethanol-induced oxidative stress in LO2 cells through MAPKs, the Keap1/Nrf2 signalling pathway and amino acid metabolism
Authors: Shi, Jie and Zhou, Xin and Zhao, Ying and Tang, Xuemei and Feng, Lu and Wang, Boyuan and Chen, Jian
Journal: Food \& Function (2021): 1672--1687

Hypocalcemia in sepsis: analysis of the subcellular distribution of Ca2+ in septic rats and LPS/TNF-$\alpha$-treated HUVECs
Authors: He, Wencheng and Huang, Lei and Luo, Hua and Zang, Yang and An, Youzhong and Zhang, Weixing
Journal: The Journal of Infection in Developing Countries (2020): 908--917

HuR regulates phospholamban expression in isoproterenol-induced cardiac remodelling
Authors: Hu, Han and Jiang, Mingyang and Cao, Yangpo and Zhang, Zhuojun and Jiang, Bin and Tian, Feng and Feng, Juan and Dou, Yali and Gorospe, Myriam and Zheng, Ming and others,
Journal: Cardiovascular research (2020): 944--955

HuR regulates phospholamban expression in isoproterenol-induced cardiac remodeling
Authors: Hu, Han and Jiang, Mingyang and Cao, Yangpo and Zhang, Zhuojun and Jiang, Bin and Tian, Feng and Feng, Juan and Dou, Yali and Gorospe, Myriam and Zheng, Ming and others, undefined
Journal: Cardiovascular research (2019)

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

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

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

EGF stimulates Mg(2+) influx in mammary epithelial cells
Authors: Trapani V, Arduini D, Luongo F, Wolf FI.
Journal: Biochem Biophys Res Commun (2014): 572

High-throughput functional assays of IP3-evoked Ca2+ release
Authors: Tovey SC, Taylor CW.
Journal: Cold Spring Harb Protoc (2013): 930

Presence of store-operated Ca2+ entry in C57BL/6J mouse ventricular myocytes and its suppression by sevoflurane
Authors: Kojima A, Kitagawa H, Omatsu-Kanbe M, Matsuura H, Nosaka S.
Journal: Br J Anaesth (2012): 352

Measurement and simulation of myoplasmic calcium transients in mouse slow-twitch muscle fibres
Authors: Hollingworth S, Kim MM, Baylor SM.
Journal: J Physiol (2012): 575

TRPC1, STIM1, and ORAI influence signal-regulated intracellular and endoplasmic reticulum calcium dynamics in human myometrial cells
Authors: Murtazina DA, Chung D, Ulloa A, Bryan E, Galan HL, Sanborn BM.
Journal: Biol Reprod (2011): 315