Magnesium Green

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MitoPedia

Magnesium Green

Description

Magnesium Green (MgG) belongs to the extrinsic fluorophores applied for measurement of mitochondrial ATP production with mitochondrial preparations. This 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.

Before the experiments, switch off the illumination in the chambers (in [Oroboros O2k] \ [O2k control], or [F7]), and set the Gain for Amp sensor to 1000 and light intensity (Amp polarization voltage [mV]) to 500.


The fluorescent dye

Magnesium GreenTM 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 of 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 Kd for magnesium is 1.0 mM, its Kd 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. To test the need to add these chelators, after addition of MgG, substrates and sample to the chamber, titrate EGTA (suggestion: 1 µM steps) until the signal no longer decreases, and then EDTA (suggestion: 1 µM steps) until the signal no longer decreases. This can be performed during the calibration and Kd determination assay. Once the concentrations of EGTA and/or EDTA to be added in the medium are determined, they can be used in the further experiments of mitochondrial ATP production with these substrates and sample.

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.

The use of creatine (e.g. MiR05Cr) is also not recommended for this technique, since it would activate creatine kinase.


Kd determination of ADP or ATP to Mg2+ using MgG

To assess the exchange of ADP/ATP by ANT using MgG, the Kd 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)

For performing these protocols, choose the following DLP files: MgG_Calibration_and_Kd_determination_ADP_Mg.DLP and MgG_Calibration_and_Kd_determination_ATP_Mg.DLP

Example of traces from calibration with MgCl2 titrations, followed by ADP/ATP titrations for determination of the Kd:

MgG Calibration and Kd determination ADP Mg.png MgG Calibration and Kd determination ATP Mg.png


Calibrating the signal and calculating the Kd of ADP or ATP to Mg2+

For performing the calibration and Kd determination, choose the following Excel template in the folder DL-Protocols\Instrumental: "Template_MgG_Calibration_and_Kd_determination_ADP_and_ATP_to_Mg".


Preparation of solutions

MgCl2 solution

Recommendation: MgCl2 solution from Sigma: M1028, 1 M solution. Prepare aliquotes before use.

Preparation of 0.1 M stock solution (dissolved in H2O):
This solution will be used for the calibration.
  1. Add 900 µL of deionized H2O in an eppendorf tube.
  2. Add 100 µL of the MgCl2 1M solution and mix.

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 εM = 15400 M-1⋅cm-1

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 εM = 15400 M-1⋅cm-1


SUIT protocol for mitochondrial ATP production and O2 flux measurements with MgG

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

For protocols with MgG, ADP must be prepared without MgCl2.


Calculating the mitochondrial ATP production

The calculation of ATP appearing in the medium is done taking in consideration the concentration of ADP added (initial concentration of ADP), the initial ATP concentration, which is considered 0 for no ATP addition, the Mg2+ concentration and the Kd of ADP and ATP for Mg2+. It is recommended to calculate both Kd values for the specific experimental conditions (the medium, substrates and sample used in the experiments).

For calculating the ATP production, choose the following Excel template in the folder DL-Protocols\SUIT: "Template - MgG ATP production analysis". A demo file is also provided.


SUITbrowser question: Mitochondrial ATP production

With the use of MgG, the mitochondrial ATP production can be assessed by high-resolution fluorespirometry.
Use the SUITbrowser to find the best protocol to answer this and other research questions.

Useful information

» A kinetic assay of mitochondrial ADP-ATP exchange rate mediated by the adenine nucleotide translocase by Chinopoulos C.
» Manual Fluorescent Magnesium Indicators.