Magnesium Green

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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+ presents 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 




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Magnesium Green in high-resolution respirometry (HRR)

Instrument

In high-resolution respirometry the MgG method is used with the O2k-FluoRespirometer with O2k-Fluo Smart-Module or O2k-Fluo LED2-Module selecting the Fluorescence-Sensor Blue/Filter Set MgG / CaG. Please see these pages for specific aspects of fluorescence measurements using the O2k.
Use black stoppers with black cover-slips to exclude disturbances by external light sources.
Before the experiments, switch off the illumination of the chambers (in [Oroboros O2k] \ [ O2k control ], or [F7]).
Set the Gain for Amp sensor to 1000 and light intensity (Amp polarization voltage [mV]) to 500. These values can be modified by the user if needed (e.g., if a different MgG concentration is used).

The fluorescent dye

Magnesium GreenTM is a registered trademark and available from Thermo Fisher Scientific (formerly: Invitrogen) in several formulations. For measuring mitochondrial ATP production a membrane impermeant formulation must be chosen (e.g. #M3733).
The technique is based on detecting the exchange of ADP/ATP by ANT. Therefore, a membrane-permeant MgG should not be used, since it might diffuse to the mitochondrial intermembrane space and matrix.

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.

Final MgG concentration in the chamber

Titration into 2 mL O2k-Chamber: 2 µL of 1.1 mM MgG stock solution, final concentration of 1.1 µM MgG.


Preparation of other solutions

MgCl2 solution

Recommendation: MgCl2 solution from Sigma: M1028, 1 M solution. Prepare aliquots 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. Aliquot (200 µL) and store at -20°C.
  5. Avoid thawing and re-freezing the aliquots.
  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. Aliquot (200 µL) and store at -20°C.
  5. Avoid thawing and re-freezing the aliquots.
  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


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 Laboratories with a modified formulation of MiR05 with 1 mM MgCl2 instead of the original 3 mM MgCl2. The medium should be prepared with the following composition: 60 mM lactobionic acid; 20 mM taurine; 10 mM KH2PO4; 20 mM HEPES; 110 mM D-Sucrose; 0.5 mM EGTA (the concentration needed can be tested previously); 1 g/L BSA (fatty acid free). The pH is adjusted with KOH to 7.1 as for MiR05-Kit, see MiPNet22.10 MiR05-kit for detailed instructions. The MgCl2 should be added only during or right before the experiments.
For the formulation of the buffer with which the method was described, see: Chinopoulos 2009 ("ANT buffer") and Chinopoulos 2014 Methods Enzymol (buffer A). It is also recommended to prepare this medium without the MgCl2, which should be added only during or right before the experiments.
For the calibration, MgCl2 is added stepwise in 0.1 mM steps, 10 times (see below, calibration and Kd determination). For experimental runs, 1 to 1.5 mM MgCl2 can be added either directly to the medium aliquot or directly to the O2k chamber before the addition of the sample. Recommendations based on the following publications:
Note that while Magnesium Green Kd for Mg2+ is 1.0 mM, Magnesium Green Kd for Ca2+ is 6 µM: this means that Magnesium Green binds more strongly to Ca2+ than to Mg2+ and therefore cannot be used in the presence of significant concentrations of free Ca2+. Possible contaminating transition metals should be chelated by a small (µM range) concentration of EGTA, EDTA or DTPA.


Titration of EGTA and EDTA
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.
Example of MgG traces where EGTA and EDTA were titrated (each event shows a titration of 1 µM EGTA or EDTA):
2018-10-03 PS3-03 - chelators titration.png


Inhibitors of ATPases and other enzymes
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.


Colour of media and chemicals used
For all fluorometric techniques, special care must be taken to avoid colored chemicals that might affect the fluorescence signal (excitation and emission wavelength spectra).
With MgG fluorophore, some uncouplers that present a yellow color might affect the signal.


Sample

The MgG technique should always be used with mitochondrial preparations, such as isolated mitochondria, permeabilized cells or tissue homogenates. This method is based on measuring ATP/ADP exchange by ANT. This requires a Mg2+-sensitive fluorescent dye, and a type of sample in which the ADP/ATP molecules are accessible to the dye, meaning that living cells cannot be used for this technique.


Calibration and 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".


SUIT protocol for mitochondrial ATP production measurement with MgG

For a SUIT protocol that uses MgG for measurement of mitochondrial ATP production combined with O2 flux measurement, see: SUIT-006 MgG mt D055, SUIT-006.
Note: For protocols with MgG dye, ADP should be prepared without MgCl2, since the technique is based on Mg2+ measurement.


Can I measure ATP production with MgG in living cells?

It is not possible to use living cells to measure ATP production with MgG, please find more information under Sample in this page.
Permeabilizing the plasma membrane with digitonin allows one to use the MgG technique with cells. The SUIT protocol SUIT-010 allows for finding the optimal digitonin concentration for different cell lines/types.


Calculating the mitochondrial ATP production

The calculation of ATP appearing in the medium is done taking into 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.

Additional information

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


Is it possible to measure ATP production by measuring only respiration?

E.g., in the SUIT-003 O2 ce D009 protocol:
In this protocol, the ROUTINE respiration of living cells is measured, which is followed by titration of oligomycin, an ATP synthase inhibitor, which will inhibit ATP production. After the oligomycin titration, LEAK respiration will be measured, and this will allow analyzing the free ROUTINE activity, the respiratory activity available for phosphorylation of ADP to ATP (see also: L/R coupling control ratio, NetROUTINE control ratio). However, this is not a direct measurement of ATP production.
A similar issue, for measurements in mitochondrial preparations in which LEAK- and OXPHOS-respiration can be measured, is discussed in the Bioenergetics Communications Mitochondrial physiology paper: "In general, it is inappropriate to use the term ATP production or ATP turnover for the difference of O2 flux measured in the OXPHOS- and LEAK-states. P-L is the upper limit of OXPHOS-capacity that is freely available for ATP production (corrected for LEAK-respiration) and is fully coupled to phosphorylation with a maximum mechanistic stoichiometry" Gnaiger et al 2020, v1.

Publications: MgG

 YearReferenceOrganismTissue;cellPreparationsStressDiseases
Salin 2019 Proc Biol Sci2019Salin K, Villasevil EM, Anderson GJ, Lamarre SG, Melanson CA, McCarthy I, Selman C, Metcalfe NB (2019) Differences in mitochondrial efficiency explain individual variation in growth performance. Proc Biol Sci 286:20191466.FishesSkeletal muscle
Liver
Homogenate
Devaux 2019 Front Physiol2019Devaux JBL, Hedges CP, Birch N, Herbert N, Renshaw GMC, Hickey AJR (2019) Acidosis maintains the function of brain mitochondria in hypoxia-tolerant triplefin fish: a strategy to survive acute hypoxic exposure? Front Physiol 9:1941.FishesNervous systemPermeabilized tissue
Isolated mitochondria
Oxidative stress;RONS
Hypoxia
Other
Salin 2018 Integr Comp Biol2018Salin K, Villasevil EM, Anderson GJ, Selman C, Chinopoulos C, Metcalfe NB (2018) The RCR and ATP/O indices can give contradictory messages about mitochondrial efficiency. Integr Comp Biol 58:486-94.FishesLiverHomogenate
Masson 2017 Sci Rep2017Masson SWC, Hedges CP, Devaux JBL, James CS, Hickey AJR (2017) Mitochondrial glycerol 3-phosphate facilitates bumblebee pre-flight thermogenesis. Sci Rep 7:13107.HexapodsSkeletal muscle
Napa 2017 Int J Dent2017Napa K, Baeder AC, Witt JE, Rayburn ST, Miller MG, Dallon BW, Gibbs JL, Wilcox SH, Winden DR, Smith JH, Reynolds PR, Bikman BT (2017) LPS from P. gingivalis negatively alters gingival cell mitochondrial bioenergetics. Int J Dent 2017:2697210.HumanEndothelial;epithelial;mesothelial cellPermeabilized cells
Salin 2016 Physiol Rep2016Salin K, Villasevil EM, Auer SK, Anderson GJ, Selman C, Metcalfe NB, Chinopoulos C (2016) Simultaneous measurement of mitochondrial respiration and ATP production in tissue homogenates and calculation of effective P/O ratios. Physiol Rep 10.14814/phy2.13007.FishesLiverHomogenate
Chinopoulos 2014 Methods Enzymol2014Chinopoulos C, Kiss G, Kawamata H, Starkov AA (2014) Measurement of ADP-ATP exchange in relation to mitochondrial transmembrane potential and oxygen consumption. Methods Enzymol 542:333-48.HumanHEKPermeabilized cells
Pham 2014 Am J Physiol2014Pham T, Loiselle D, Power A, Hickey AJ (2014) Mitochondrial inefficiencies and anoxic ATP hydrolysis capacities in diabetic rat heart. Am J Physiol 307:C499–507.RatHeartHomogenateIschemia-reperfusion
Oxidative stress;RONS
Mitochondrial disease
Diabetes
Myopathy
Power 2014 Physiol Rep2014Power A, Pearson N, Pham T, Cheung C, Phillips A, Hickey A (2014) Uncoupling of oxidative phosphorylation and ATP synthase reversal within the hyperthermic heart. Physiol Rep pii:e12138.RatHeartIsolated mitochondriaTemperature
Goo 2013 Clin Exp Pharmacol Physiol2013Goo S, Pham T, Han JC, Nielsen P, Taberner A, Hickey A, Loiselle D (2013) Multiscale measurement of cardiac energetics. Clin Exp Pharmacol Physiol 40:671-81.HeartPermeabilized cells
Homogenate
Isolated mitochondria
Oxidative stress;RONS
Iftikar 2013 PLoS One2013Iftikar FI, Hickey AJ (2013) Do mitochondria limit hot fish hearts? Understanding the role of mitochondrial function with heat stress in Notolabrus celidotus. PLoS One 8:e64120.FishesHeartPermeabilized tissueOxidative stress;RONS
Chinopoulos 20092009Chinopoulos Christos, Vajda Szilvia, Csanady Laszlo, Mandi Miklos, Mathe Katalin, Adam-Vizi Vera (2009) A Novel Kinetic Assay of Mitochondrial ATP-ADP Exchange Rate Mediated by the ANT. Biophys J 96, 2490-504.RatHeart
Nervous system
Liver
Isolated mitochondria
Abstracts: MgG