MiPNet20.14 AmplexRed H2O2-production

O2k-Fluorometry: HRR and simultaneous determination of H2O2 production with Amplex Red.

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OROBOROS (2015-06-15) Mitochondr Physiol Network

Abstract: Krumschnabel G, Fontana-Ayoub M, Fasching M, Gnaiger E (2015) O2k-Fluorometry: HRR and simultaneous determination of H₂O₂ production with Amplex Red. Mitochondr Physiol Network 20.14(01):1-6.

High-resolution respirometry [1] is extended by O2k-MultiSensor modules allowing the simultaneous measurement of oxygen consumption and one additional parameter (mitochondrial membrane potential, pH, Ca²⁺, or NO concentration [2-5]). Fluorometric methods are available for a wide range of analytical parameters of major interest in mitochochondrial physiology: H₂O₂ production, mitochondrial membrane potential, intracellular pH, Ca²⁺, Mg²⁺, and NADH levels. Advantages of the simultaneous measurement of additional parameters in a single respirometric chamber:

1. Quality control of preparations by respirometric performance.
2. Objective exclusion criteria for protocol related problems, e.g. overtitration of uncouplers, side effects of TPP⁺ or fluorophores.
3. Elimination of artifacts of normalization for a mitochondrial marker when combining two methods in a single chamber.
4. Additional information is acquired for a limited amount of sample.
5. Oxygen dependence of parameters can be studied.

In the publication (Krumschnabel et al 2015) we introduce the O2k-Fluo LED2-Module, describe one selected stage in its development, and show its application for the simultaneous measurement of respiration and H₂O₂ production of mouse brain mitochondria.

Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. »Bioblast link«

O2k-Publication: »Makrecka-Kuka 2015 Biomolecules

• O2k-Network Lab: AT_Innsbruck_OROBOROS

Labels: MiParea: Respiration, Instruments;methods 

Organism: Mouse  Tissue;cell: Heart  Preparation: Isolated mitochondria 

Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property. 

HRR: Oxygraph-2k, O2k-Fluorometer, O2k-Protocol 

O2k-Demo, O2k-MultiSensor, MitoFitPublication 

Applications with O2k-Fluorometry

The Amplex Red method is used with the O2k-Fluo LED2-Module selecting the Fluorescence-Sensor Green. Use black stoppers with black cover-slips to exclude disturbances by external light sources.

The fluorescence sensors are inserted though the windows of the O2k-Chambers. The O2k has also been coupled to a fluorescence spectrophotometer with a light guide inserted through the black PEEK stopper.

Preparation of Amplex® UltraRed solutions

Source: Life Technologies (former Invitrogen) A36006

See the manual provided by the producer: Manual Amplex UltraRed.

Storage solution = stock solution

Prepare a 10 mM storage solution of Amplex® UltraRed reagent (AmR), which will also serve as stock solution, by adding 340 μL of fresh, high‑quality DMSO (Sigma D8418) to one commercial vial of Amplex® UltraRed reagent (1 mg). Vortex well to dissolve. Protect AmR from light and moisture. The storage solution should then be divided into 10 µl aliquots and stored in the dark, protected from moisture at –20°C for future use. When stored properly, the 10 mM storage solution is stable for at least 6 months.

Note: The preparation of the 10 mM storage solution follows the procedure suggested by the manufacturer. The manufacturer states only an approximate molecular weight for the Amplex® UltraRed formulation and does not publish details how the Amplex® UltraRed formulation deviates from the substance 10-acetyl-3,7-dihydroxyphenoxazine (CAS# 119171-73-2), known as Amplex® Red.

Final AmR concentration

• Titration into into 2 ml O2k-chamber: 2 µl of 10 mM AmR stock solution, final concentration of 10 µM AmR.

You may add 1 µl of the 10 mM stock solution for brief measurements to obtain a final concentration of 5 µM. Optimize the final AmR concentration according to respiration media, sample type and concentration, and experimental protocol. These determine the total AmR consumption by H₂O₂ production during the experiment (check by initial and final H₂O₂ titrations for calibration). The final concentration of AmR becomes diminished during an experiment due to AmR consumption and dilution by titrations in a SUIT protocol. Check for any potential effects of the exprimental AmR concentration on respiratory activity of cells or mt-preparations, by titration of AmR after a specific respiratory state has been reached in a control experiment.

Horse radish peroxidase

• Titration into into 2 ml O2k-chamber: 4 µl of HRP stock solution, final concentration 1 U/ml.

Horse radish peroxidase (Sigma-Aldrich P 8250 - 5 kU): Prepare a stock solution with 500 U HRP/ml in MiR05 or MiR05Cr; the solution can be used as storage solution at -20 °C.

Superoxide dismutase

Superoxide dismutase (SOD) is optionally included to generate H₂O₂ from superoxide. Results may be compared with and without SOD for evaluation of the contribution of superoxide not endogenously dismutated with the formation of peroxide.

(Sigma-Aldrich S 8409-15KU): Depending on the batch the SOD preparation may contain a specific activity of 2,000–6,000 units/mg protein. We recommend to use the enzyme at 5 U/ml, the final volume to be added to the respiratory chamber has thus to be adjusted accordingly.

Calibration with H₂O₂

Calibration standards of H₂O₂: Commercial solution (Sigma-Aldrich 323381 - 25 ml hydrogen peroxide solution 3 wt. %; stabilized with acetanilide, c. 200 ppm) = 880 mM H₂O₂.

1. To prepare a HCl stock solution of 10 µM HCl, take 1 ml of 1 mM HCl storage solution an fill up to 100 ml with distilled water.
2. H₂O₂ dilution 1 (1:88): add 284 µl of a commercial solution of 3 wt.% H₂O₂ to 20 ml of 10 µM HCl and fill up to a final volume of 25 ml with 10 µM HCl, to obtain a 10 mM H₂O₂ solution.
3. H₂O₂ dilution 2 (1:250): dilute 200 µl of 10 mM H₂O₂ solution with 10 µM HCl to a volume of 50 ml to obtain the H₂O₂ stock solution of 40 µM.

• Titration into into 2 ml O2k-Chamber: 5 µl of the 40 µM H₂O₂ stock, step increase of 0.1 µM H₂O₂. One to three subsequent steps.

Titrations are peformed after addition of respiration medium, HRP and Amplex UltraRed to the O2k chamber. We suggest to perform calibrations at the beginning (sample already present), intermittently at various respiratory states, and near the end of an experiment (but before cytochrome c, catalase or other substances are added that are interfer with the H₂O₂ measurement).

DatLab supports automatic H₂O₂ calibration by calculating the calibration parameters by linear regression and graphical display of the calibration regression:

>> MiPNet19.18D_O2k-Calibration.

Since part of the signal change might be caused by an ongoing H₂O₂ production of the biological sample the calibration can be corrected by including the slope in the calculation.

DatLab 6

The slope used for the correction is calculated from a linear regression of the signal vs time for the selected marks. This eliminates the distortion of the slope by injection artifacts after the Amp signal is already stable.

• Select/deselect slopes to be include in the calculation by ticking the check boxes on the right of the slope values. If you select more than one slope the weighted mean of all values is used for calculation.

Substances incompatible with the Amplex Red method

The following substances/ classes of substances are strictly incompatible with the Amplex Red method for theoretical reasons:

• Strongly redox active substances, e.g. cytochrome c, TMPD/Ascorbate

• Inhibitors of horseradish peroxidase, e.g. cyanide, azide; more information: horseradish peroxidase

• Catalase and other substances consuming or scavanging H₂O₂. The effect of substances in the medium that consume H₂O₂ slowly is taken account of by the calibration procedure. However, such substances decrease the sensitivity of the method. Note that catalase can be a valuable tool for checking for artifacts.

The effect of other substances should be checked by blank experiments, including comparing the sensitivity (result of calibration) before and after injecting the substances.

In preliminary experiments OROBOROS INSTRUMENTS evaluated small amounts (as typically used in SUIT protocols) of the following substances as compatible with the method:

DMSO, ethanol, malate, glutamate, pyruvate (a strong scavanger of H₂O₂), succinate, (ADP + Mg²⁺), (ATP + Mg²⁺), rotenone, FCCP, oligomycin, antimycin A, malonate, myxothiazol

Checking for artifacts

The Amplex method is based on the H₂O₂ dependent oxidation of AmR to resorufin by HRP. Under unfavorable conditions AmR may be oxidized even in the absence of H₂O₂. At a small rate such an oxidation occurs in the presence of HRP even without any sample present. The magnitude of this background signal ("drift") depends, among other things, on the light intensity used and can therefore be minimized by using the suggested or lower light intensity. Components of the sample may however induce a far higher, non H₂O₂ related rate of AmR oxidation. Therefore, especially when applying the method on new types of samples the method should be checked for artifacts. A few approaches are listed here:

Sequential addition of HRP and AmR: This method is particular easy to implement if AmR and HRP have to be added to the chamber already containing the sample anyway: First, inject Amplex Red and wait a few minutes for flux stabilization. The Amp slope has to stay near zero. Then add HRP. The Amp slope should increase and correspond to the H₂O₂ production. If a significant Amp slope is detected before the addition of HRP this increase in fluorescence is not caused by H₂O₂ production. The experiment can be continued as usual after this test. If the sample is injected routinely into the chamber already containing AmR and HRP the method can not be applied. In this case it is suggested to change this sequence at least for one experiment.

Addition of catalse: After a H₂O₂ flux (production) is established, a high dose (e.g. 10 µl of a 280,000 U/ml stock solution) of catalase is injected. Catalase competes with HRP for the available H₂O₂. Then the apparent H₂O₂ production (the Amp slope) should be reduced to near zero as a control for distinguishing an unspecific chemical background slope from H₂O₂ dependent Amplex Red oxidation.

Note: The experiment cannot be continued afterwards for measurement of hydrogen peroxide production, whereas respiration can be recorded onwards.