Amplex UltraRed

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Amplex UltraRed

Description

Amplex® UltraRed (AmR) is used as an extrinsic fluorophore for measurement of hydrogen peroxide production (ROS) by cells or mitochondrial preparations. The reaction of H2O2 and AmR is catalyzed by horseradish peroxidase to produce the red fluorescent compound resorufin (excitation wavelength 563 nm, emission 587 nm; the fluorescent product according to the supplier is called UltroxRed in the case of Amplex® UltraRed which has a very similar structure to resorufin). The change of emitted fluorescence intensity is directly proportional to the concentration of H2O2 added, whereby the H2O2 is consumed.

Abbreviation: AmR

Reference: Mohanty 1997 J Immunol Methods, Zhou 1997 Anal Biochem, Mishin 2010 Free Radical Biol Med, Towne 2004 Anal Biochem, Krumschnabel 2015 Methods Mol Biol



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Amplex Red or Amplex UltraRed in research articles

Citation Amplex HRP pH Limit of detection
stock final unit definition stock final
mM
µM
U/ml
U/ml
BIOTEK
10
50
pyrogallol
10
0.1
7.4
4 nM (absorption 300 nm)
Life Technologies
10
50
10
0.1
6 to 7.5
<80 nM
Towne 2004
160
0.41
7.5 to 8.5
100 nM
Zhou1997
3 ?
0.3 to 1
50 nM (10 nM optimal)
Mohanty 1997
100
10 to 100
1
18 nM
Komary 2010
1
2.5


Amplex® UltraRed in O2k-FluoRespirometry

O2k-Fluo Smart-Module and O2k-Fluo LED2-Module

In high-resolution respirometry HRR the Amplex® UltraRed method[1] is used with the O2k-FluoRespirometer selecting the Fluorescence-Sensor Green (O2k-Series D-G: O2k-Fluo LED2-Module) and Smart Fluo-Sensor for O2k-Series H - I. 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[2]. The O2k has also been coupled to a fluorescence spectrophotometer with a light guide inserted through the black PEEK stopper.[3]
The use of AmR in HRR to simultaneously determine respiration and H2O2 flux has been intensively tested in the Oroboros lab (see references).

Aplication in HRR

Amplex® UltraRed

AmR: Amplex® UltraRed from Life Technologies (former Invitrogen): A36006; 6* 1 mg commercial vials, store at -20°C.
Preparation of 10 mM stock solution (dissolved in DMSO) - see also Manual Amplex® UltraRed by Life Technologies.
Storage solution = stock solution
Prepare a 10 mM storage solution of Amplex® UltraRed reagent (AmR), which will also serve as a 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 20 µL aliquots and stored in the dark in a desiccator, protected from moisture at –20°C for future use. When stored properly, the 10 mM storage solution is stable for 6 months. We have observed that after long-term storage AmR may exert a profound inhibitory effect on cell respiration.
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.
» O2k manual titrations MiPNet09.12 O2k-Titrations
Titration into 2 mL O2k-chamber: 2 µL of 10 mM AmR stock solution, final concentration of 10 µM AmR.
AmR, similar to other chemical probes, may exert an inhibitory effect on mitochondrial and cell respiration. AmR, therefore, should be used at the lowest concentration compatible with the total H2O2 production in an experimental run, including calibrations and chemical background conversion of AmR [4],[5].
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 experimental 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.

Color of AmR and the medium

Amplex UltraRed freshly dissolved should not present any color. The appearance of pink coloring may indicate that the chemical has been compromised.
During the experimental run, the product UltroxRed (or resorufin), generated from the Amplex UltraRed (or Amplex Red) plus H2O2 in the reaction catalyzed by horseradish peroxidase, accumulates in the chamber. As this chemical has a pink color, it is normal for the medium to have a pink color at the end of the experiment.

Horseradish 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 included to generate H2O2 from superoxide.
(Sigma-Aldrich S8160-15KU): 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.
Experiments can be performed in the absence of SOD.

DTPA

-See:DTPA

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. Prepare a HCl stock solution of 10 µM HCl.
  2. H2O2 dilution 1 (1:88): add 10 µL of a commercial stock solution of 3 wt. H2O2 to 870 µL 10 µM HCl solution, to obtain a 10 mM H2O2 solution.
  3. H2O2 dilution 2 (1:250):add 4 µL of 10 mM H2O2 solution to 996 µL 10 µM HCl solution, to obtain the H2O2 stock solution of 40 µM.
The H2O2 calibration solution has to be prepared fresh every day, kept away from light exposure, and freezing should be avoided.
  • Titration into 2 mL O2k-chamber: 5 µL of the 40 µM H2O2 stock, step increase of 0.1 µM H2O2.
  • Background H2O2 calibration:
  • A set of H2O2 titrations conducted after the addition of respiration medium, (DTPA), HRP and Amplex® UltraRed to the O2k-chamber but before sample addition.
  • This step is required (1) for the calculation of chemical background fluorescence; see: MiPNet24.10 H2O2 flux analysis and How to analyze H2O2 flux in DatLab, and (2) for calculation of the sensitivity of the Amplex® UltraRed assay towards H2O2, which, therefore, serves as a quality control step. The sensitivity depends on the respiration medium and on the components of the Amplex® UltraRed assay, and in the same respiration medium, the sensitivity of the AmR assay should be in the same range. For more information: Komlodi 2018 Methods Mol Biol.
  • Background H2O2 calibration has to be performed for each individual instrumental setting (O2k, fluorescence sensor and chamber), if a new stock solution of DTPA, HRP, SOD, Amplex® UltraRed, or H2O2, is prepared, or a different respiration medium is used. As the H2O2 stock solution needs to be freshly prepared, it is recommended to perform a background H2O2 calibration before each experiment and at a minimum once per experimental day.
  • The protocol for background H2O2 calibration is provided with DatLab7.4. For further information, see: Instrumental: Browse DL-Protocols and templates
  • Afterwards we suggest to perform calibrations at the beginning (sample already present), intermittently at various respiratory states, and near the end of an experiment. These calibrations steps are recommended because over the experimental time the sensitivity decreases and chemicals can influence the sensitivity of the AmR assay towards H2O2. After the addition of the biological sample, the sensitivity usually decreases owing to the (high) antioxidant capacity of the sample. For further information, see: Komlodi 2018 Methods Mol Biol


  • Why it is necessary to perform multiple H2O2 calibrations in the Amplex® UltraRed (AmR) assay?
The sensitivity to H2O2 of the Amplex® UltraRed assay changes during the experiment, thus requires multiple calibrations. These sensitivity changes may be the function of (1) experimental time and accumulating UltroxRed (similar to resorufin), (2) changes of the optical properties due to titrations, (3) the radical scavenging capacity of the sample. Therefore, H2O2 calibrations are performed before and after sample addition, and after selected titration steps. - »Komlodi 2018 Methods Mol Biol«
The DLPs for AmR SUIT protocols already come with multiple steps of H2O2 calibrations implemented.- » SUITbrowser«
  • DatLab supports automatic H2O2 calibration by calculating the calibration parameters by linear regression and graphical display of the calibration regression.
Problem: Here is a DatLab file where I ran the H2O2 calibration protocol with 6 H2O2 titrations. I drew up 5 uL of H2O2 into the syringe for titration into each chamber individually for the first two titrations. For the remaining titrations, I drew up 10 uL of H2O2 into the syringe and added 5 uL to one chamber and the rest into the second chamber. For titrations 3 and 4 I added to chamber B first, followed by chamber A. For titrations 5 and 6 I added to chamber A first, followed by chamber B. If sensitivities are measured for each set of titrations based on the manner in which H2O2 was added to the chambers, it can be seen that the chamber which is titrated second when 10 uL of H2O2 is drawn into the syringe at one time has reduced sensitivity measurements. When fresh H2O2 is drawn up for each chamber individually, the sensitivities between chambers are much more comparable. (The numbers shown in the figures are sensitivities expressed as V/µM.)
Jennifer Norman 1.png Jennifer Norman 2.png
Answer: It is recommended to fill the syringe with H2O2 each time with 5 µL for each O2k-Chamber instead of filling up the syringe with 10 µL and titrate one chamber after the other. Based on tests of Jennifer Norman (US_CA Davis_Roshanravan B) it seems that either the syringe for H2O2 titration is not precise filling it up with the maximal volume or H2O2 might be degraded by light already in the syringe.




Experimental media for the AmR assay

Media with high antioxidant activity compete with HRP and partially consume H2O2 before it can react with AmR to form the active fluorophore UltroxRed (similar to resorufin). This was shown by comparing the resorufin-sensitivity and H2O2-sensitivity [6],[7].
  • H2O2-sensitivity: the change of fluorescence signal per µM H2O2 added in calibrations with H2O2 titration in media containing HRP and AmR.
  • Resorufin-sensitivity: the change of fluorescence signal per µM resorufin added in calibrations with resorufin titration in media containing HRP and AmR.
The H2O2-sensitivity is much higher in a simple phosphate buffer compared to media with strong antioxidant capacity. In contrast, this is not the case for the resorufin-sensitivity.

H2O2 sensitivity in MiR05

We recommend running experiments in MiR05 with a sensitivity >0.5 and <3 at gain 1000 and fluorescence intensity lower than 500 . If the sensitivity is out of this range, please change the light intensity of the Fluo-sensors (in DatLab 7.4: [Oroboros O2k] \ [O2k control], click on the tab [Amperometric, Amp] and change light intensity (Amp polarization voltage [mV]). Do the calibration again by titrating H2O2 to check the sensitivity.
The sensitivity values may vary depending on the batch of medium and chemicals used.
The sensitivity value is used to calibrate the amperometric raw signal [V] to H2O2 equivalent concentration [µM]. If experiments present different sensitivities, the resulting H2O2 fluxes after calibration can still be comparable, as long as the sensitivity values are in the recommended range and the same settings are used for all experiments.

Calculation of background fluorescence slope of MiR05

In the absence of sample, there is a spontaneous increase of the Ultroxred fluorescence signal over time, the extent of which depends on components of the respiration medium (Krumschnabel et al 2015). The sensitivity – the change in fluorescence per unit of H2O2 produced in or added to the chamber – depends on the medium and tends to decline over time. Both the background change in fluorescence and the change in assay sensitivity over time need to be corrected for in data analysis, for which DatLab-Analysis templates are available.
  • The Excel template is ideal for analysis of O2 and H2O2 fluxes measured in MiR05-Kit supplemented with DTPA. The background fluorescence slope of the AmR assay is dependent on the Lot number of the MiR05-Kit, therefore, in each SUIT-###_AmR folder, in the Excel templates for MiR05-Kit (supplemented with DTPA) you can find different analysis templates for each Lot number. The Excel sheets differ from each other in the correction for the background fluorescence slope. You need to use the Excel template which corresponds with the Lot# of MiR05-Kit you use. If you do not add DTPA or use homemade MiR05 instead of the MiR05-Kit, we recommend calculating the background fluorescence slope. The calculation of the background fluorescence slope is also advisable when a new batch of Amplex UltraRed is used or a newly prepared MiR05 is applied. Please follow the steps to measure and calculate background fluorescence slope:
a. Go to [Layout] menu and click into ‘O2&Amp´ and select `Standard Layouts/01 Amp Amperometric_Raw signal´.
b. Go to `Marks´ and select ‘Slope uncorrected + all info´. In the new window select `AmR slope [pmol/s*mL]´ in `Plot for Marks´.
c. Channel: `Amperometric,Amp´. Leave only this channel selected.
d. Select: `Median´.
e. Sort by: `Time´(default).
f. Then, click on [Copy to clipboard] to copy the selected values.
  • In the Amp background fluorescence slope.xlsx Excel template (see below): Click on the yellow cell A1 and paste [Ctrl+V] Amp slope from DatLab. If more than one experiment is performed, copy the Amp slope on A32, A63, A94, A126 or A158.
  • In the "Data" sheet, write the number of the FluoSensors in cells B12. If more than one sensor was tested, write the sensor number in cells B43, B74, B105, B137 or B169. The numbers can be found in the O2k control window (select in ´Oroboros O2k´ menu or press F7), ´Amperometric, Amp´ tab.
  • Equation required for the correction for the background fluorescence slope using the applied FluoSensor can be found in the figure. The values can be also found in the cells H18 and I18, H48 and I48, H79 and I79, H110 and I110, H142 and I42 or H174 and I174.
  • It is advisable to measure thebackground fluorescence slope with the same FluoSensors in the same O2k-chambers several times and then calculate the plot of the equation from more experiments. In the Excel template in the ´Summary and equation´ tab, you can copy a° and b° into the table to calculate the equation of plot.
  • In the SUIT-###_AmR analysis templates, modify the values in the equation for the background fluorescence slope (cells N66, W66, and X66).


HRP-independent background AmR flux

A study by Miwa et al 2016 (Free Radical Bio Med 90:173) suggests that the HRP-independent artificial background is related to a mitochondrially expressed carboxylesterase which converts Amplex® Red to resorufin at a high rate. The issue can be relatively easily solved by adding a protease inhibitor to various mitochondrial preparations as described here.
For further information see the Discussion page.

AmR assay dilution

How to minimize dilution of the AmR assay upon sample addition
To minimize the dilution of the AmR assay either:
1.) Decrease the titration volume of the sample by using a more concentrated sample solution
2.) Titrate additionally the components of the AmR assay together with the sample
3.) After AmR background calibration, empty and wash the chamber, then add buffer and sample and start titrating the components of the AmR assay into the closed O2k-chamber.
In either case 2 or 3, use media and chemicals from the same batch.
Of note, performing H2O2 calibration directly after sample addition allows for calculating the sensitivity to correct the fluorescence signal for sample dilution.

Artefacts

Liver homogenate

Liver homogenate cannot be used with the Amplex® UltraRed assay. - See:Tissue homogenate

Injection artefact

Injection of chemicals (e.g. substrates, uncouplers, inhibitors, H2O2) can cause artefacts as observed in the figure:
Injection artefact AmR.png
The marks should always be set on the stable signal avoiding the artefacts area.
Injection artefact AmR marks.png

NADH and reduced glutathione

Votyakova TV, Reynolds IJ (2014 Arch Biochem Biophys 431:138-44) showed that NADH and reduced glutathione are able to react with AmR in the presence of O2 and HRP and create a background in the absence of mitochondria. If the mitochondria remain intact, this above-mentioned reaction is negligible. If you add SOD (superoxide dismutase), you can avoid this side-effect of the AmR assay.

Substances incompatible with the Amplex® UltraRed assay

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


The effect of other substances should be checked by experiments without biological sample, 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, CCCP, oligomycin, antimycin A, malonate, myxothiazol

Avoiding artefacts

The Amplex® method is based on the H2O2 dependent oxidation of AmR to UltroxRed 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 catalase: 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 flux (the Amp slope) should be reduced to near zero as a control for distinguishing an unspecific chemical background slope from H2O2 dependent Amplex® UltraRed oxidation.
Note: The experiment cannot be continued afterwards for measurement of hydrogen peroxide production, whereas respiration can be recorded onwards.

Nitrogen injection

Experimental SOP

Permeabilized fibers

Why are permeabilized muscle fibers (pfi) a poor sample type for studying reactive oxygen species (ROS) production?
In experiments with pfi, high oxygen concentrations are needed to avoid oxygen limitation. However, the high oxygen pressures used may artificially increase ROS, including H2O2 production, making pfi a less optimal model for ROS production studies. See:Oxygen dependence of pfi

H2O2 flux analysis and mark setting

Excel analysis templates

  • Excel templates are provided for analysis of the H2O2 measurements. It can be found in the upper menu in DatLab in Protocols/SUIT: Browse DL-Protocols and templates and then select your protocol (e.g. SUIT-009/SUIT-009_AmR/SUIT-009_AmR_ce-pce_D019).
  • The calculations used in the excel analysis template are provided complying with Oroboros transparency policy: [1]
  • The excel analysis templates are updated for the respective SUIT protocol. The latest versions are available here (last update 2021-09-20):

SUIT protocols

Troubleshooting

How to set a mark to calibrate the fluorescence signal using Amplex® UltraRed assay?

To calibrate the fluorescence signal into µM, set the marks to the black line.
  • -See:
MiPNet20.14 AmplexRed H2O2-production
Amp calibration - DatLab

How to set a mark to analyse H2O2 flux?

  • -See:
MiPNet20.14 AmplexRed H2O2-production
Smoothing
H2O2 flux analysis video

How to analyse the data in the Excel template?

  • -See:
MiPNet24.10 H2O2 flux analysis
H2O2 analysis
H2O2 flux analysis video

Defective fluorescence module

How to exchange the filters in the fluorescence sensors?

How to connect the Smart Fluo-Sensors to the O2k?

References

  1. Mohanty 1997 J Immunol Methods
  2. Hickey 2012 J Comp Physiol B
  3. Anderson 2011 Am J Physiol Heart Circ Physiol
  4. Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38. - »Bioblast link«
  5. Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55. - »Bioblast link«
  6. 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«
  7. Komlodi T, Sobotka O, Krumschnabel G, Bezuidenhout N, Hiller E, Doerrier C, Gnaiger E (2018) Comparison of mitochondrial incubation media for measurement of respiration and hydrogen peroxide production. Methods Mol Biol 1782:137-55. - »Bioblast link«
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