Magnesium Green

high-resolution terminology - matching measurements at high-resolution

Magnesium Green

Description

Magnesium Green(MgG) belongs to the extrinsic fluorophores applied for measurement of mitochondrial ATP production with mitochondrial preparations. This fluorescent dye fluoresces when bound to Mg²⁺. The technique to measure mitochondrial ATP production is based on the fact that Mg²⁺ present different dissociation constants for ADP and ATP, and the adenine nucleotide translocase (ANT) exchanges ATP for ADP.

Abbreviation: MgG

Reference: Chinopoulos 2014 Methods Enzymol

MitoPedia methods: Fluorometry 

Magnesium Green in high-resolution respirometry (HRR)

Instrument

In high-resolution respirometry the MgG method is used with the O2k-FluoRespirometer with O2k-Fluo LED2-Module) selecting the Fluorescence-Sensor Blue/Filter Set MgG / CaG. Use black stoppers with black cover-slips to exclude disturbances by external light sources.

The fluorescent dye

Magnesium Green^TM is a registered trademark and available from Thermo Fisher Scientific (former: Invitrogen) in several formulations. For measuring mitochondrial ATP production a membrane impermeant formulation must be chosen (e.g. #M3733).

Preparation of MgG solution

Magnesium Green from Thermo Fischer Scientific (former Invitrogen): M3733 (Magnesium Green™, Pentapotassium Salt, cell impermeant); 1 mg vial, store at -20°C.

Preparation of 1.1 mM stock solution (dissolved in H₂O):

1. Dissolve the complete vial of MgG (1 mg) in 992.6 µL of deionized H₂O.
2. Divide into 40 µL portions into 0.2 mL Eppendorf tubes (protect from light, use dark tubes preferably).
3. Store frozen at -20 °C protected from light.

Experimental media for the MgG assay

This method is not suitable for buffers containing high concentrations og Mg²⁺. It has been used in the OROBOROS Mitochondrial Research Laboratory with a modified formulation of MiR05 with 1 mM MgCl₂ instead of 3 mM MgCl₂. For the formulation of the buffer with which the method was described, see: Chinopoulos 2014 Methods Enzymol.

Note that while MgG K_D for magnesium is 1.0 mM, its K_D for calcium is 6 µM: Magnesium Green binds stronger to calcium than to magnesium and cannot be used in the presence of significant concentrations of free calcium. Possible contaminations with transition metals should be complexed by a small (µM range) concentration of EGTA, EDTA or DTPA.

For samples that present contamination by ATPases and other ATP-consuming enzymes, the use of inhibitors such as sodium orthovanadate and beryllium fluoride is recommended (Chinopoulos et al., 2014). Pham et al., 2014 used blebbistatin to inhibit myosin heavy chain and ouabain to inhibit Na+,K+-ATPase with rat heart homogenates.

K_d determination of ADP or ATP to Mg²⁺ using MgG

To assess the exchange of ADP/ATP by ANT using MgG, the K_d of ADP and ATP to Mg²⁺ should be previously calculated for the pertaining experimental conditions. This can be done in the O2k FluoRespirometer with a protocol in which a series of MgCl₂ titrations are performed for calibration up to 1 mM MgCl2, and then a series of titrations with ADP or ATP are performed.

1. Add to the chamber the same respiration media that will be used for experiments, without MgCl₂.
2. Add MgG to the chamber and allow stabilization of the signal if necessary. It is also possible to dilute MgG in the respiration medium prior to addition to the chamber
3. Add carboxyatractyloside to inhibit the transport of ADP/ATP, oligomycin to inhibit ATP-synthase activity and Ap5A to inhibit adenylate kinase.
4. Add to the chambers the same substrates and sample to be used in the experiments.
5. Titrate MgCl₂ to the chamber. 10 titrations in 0.1 mM steps are recommended (2 µL of 0.1 mM solution).
6. Titrate ADP or ATP to the chamber. This must be done separately in two chambers, one for ADP and one for ATP.

• ADP: 0.2 M stock solution, 19 titrations with 2.5 µL (0.25 mM per titration)
• ATP: 0.2 M stock solution, 11 titrations with 2 µL (0.2 mM per titration)

Recommendations: MgCl₂: Sigma M1028

Preparation of ADP solution

ADP from Merck (former Calbiochem): 117105 (Adenosine 5ʹ-Diphosphate, Potassium Salt), store at -20°C.

Preparation of 0.2 M stock solution (dissolved in H₂O):

1. Weigh 1.0026 g, dilute in H₂O
2. Adjust pH to 6.9 with KOH, preferably on ice (with pHmeter calibrated on the same condition)
3. Complete with H₂O to 10 mL
4. Aliquote (200 µL) and store at -20°C.
5. Avoid thawing and re-freezing the aliquotes.
6. The concentration can be corrected by measuring the absorbance at 260 nm and using an extinction coefficient factor of eM = 15400 M^-1cm^-1

Preparation of ATP solution

ATP from Merck (former Sigma-Aldrich): A26209 (Adenosine 5′-triphosphate disodium salt hydrate), store at -20°C.

Preparation of 0.2 M stock solution (dissolved in H₂O):

1. Weigh 1.1023 g, dilute in H₂O
2. Adjust pH to 6.9 with KOH, preferably on ice (with pHmeter calibrated on the same condition)
3. Complete with H₂O to 10 mL
4. Aliquote (200 µL) and store at -20°C.
5. Avoid thawing and re-freezing the aliquotes.
6. The concentration can be corrected by measuring the absorbance at 260 nm and using an extinction coefficient factor of eM = 15400 M^-1cm^-1

SUIT protocol with MgG

For a SUIT protocol with MgG for measurement of mitochondrial ATP production, see: SUIT-006 MgG mt D055, SUIT-006.

Useful information

» A kinetic assay of mitochondrial ADP-ATP exchange rate mediated by the adenine nucleotide translocase by [Chinopoulos C]

» Manual Fluorescent Magnesium Indicators.