View source for Amplex UltraRed

{{MitoPedia
|abbr=AmR
|description='''Amplex UltraRed''' (AmR) is used as an [[extrinsic fluorophores |extrinsic fluorophore]] for measurement of [[hydrogen peroxide]] production ([[ROS]]) by cells or mitochondrial preparations. The reaction of H<sub>2</sub>O<sub>2</sub> and AmR is catalyzed by [[horseradish peroxidase]] to produce the red fluorescent compound [[resorufin]] (excitation wavelength 563 nm, emission 587 nm;  the fluorescenet 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 H<sub>2</sub>O<sub>2</sub> added, whereby the H<sub>2</sub>O<sub>2</sub> is consumed.
|info=[[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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{{Technical support integrated}}

=== Sources of AmR ===

{| style="border-spacing:0;"
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| '''Trade Mark'''
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| '''Manufacturer/ Distributor'''
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">'''<nowiki>price [€/mg]</nowiki>'''</div>
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">'''product id'''</div>
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.5cm;padding-right:0.053cm;"| <div align="left"> '''Additional information'''</div>

|-
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| Amplex Red 
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <center>Life Technologies</center>
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">37.4</div>
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">A12222</div>
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.5cm;padding-right:0.053cm;"| <div align="left"> https://www.thermofisher.com/order/catalog/product/A12222</div>

|-
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| Amplex Ultra Red
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <center>Life Technologies</center>
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">48.8</div>
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">A36006</div>
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.5cm;padding-right:0.053cm;"| <div align="left"> https://tools.thermofisher.com/content/sfs/manuals/mp36006.pdf</div>

|-
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| Ampliflu Red 
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <center>Sigma</center>
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">18.5</div>
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">90101</div>

|-
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| Quanta Red 
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <center>Thermo scientific</center>
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| 
| style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">15150</div>

|}


{|  border="1"  style="border-spacing:0;" style="text-align: center;"
|| Citation
| colspan="2" |  Amplex
| colspan="3" | HRP
|| pH
|| Limit of detection

|-
|| 
|| stock
|| final 
|| unit definition
|| stock
|| final
|| 
|| 

|-
|| 
|| <div align="right">mM</div>
|| <div align="right">µM</div>
|| 
|| <div align="right">U/ml</div>
|| <div align="right">U/ml</div>
|| 
|| 

|-
|| BIOTEK
|| <div align="right">10</div>
|| <div align="right">50</div>
|| <div align="right">pyrogallol</div>
|| <div align="right">10</div>
|| <div align="right">0.1</div>
|| <div align="right">7.4</div>
|| <div align="right">4 nM (absorption 300 nm)</div>

|-
|| Life Technologies
|| <div align="right">10</div>
|| <div align="right">50</div>
|| 
|| <div align="right">10</div>
|| <div align="right">0.1</div>
|| <div align="right">6 to 7.5</div>
|| <div align="right"><nowiki><80 nM</nowiki></div>

|-
|| [[Towne_2004_Anal_Biochem|Towne 2004]]
|| 
|| <div align="right">160</div>
|| 
|| 
|| <div align="right">0.41</div>
|| <div align="right">7.5 to 8.5</div>
|| <div align="right">100 nM</div>

|-
|| [[Zhou_1997_Anal_Biochem|Zhou1997]]
|| 
|| <div align="right">3 ?</div>
|| 
|| 
|| <div align="right">0.3 to 1</div>
|| 
|| <div align="right">50 nM (10 nM optimal)</div>

|-
|| [[Mohanty_1997_J_Immunol_Methods |Mohanty 1997]]
|| <div align="right">100</div>
|| <div align="right">10 to 100</div>
|| 
|| 
|| <div align="right">1</div>
|| 
|| <div align="right">18 nM</div>

|-
|| [[Komary 2010 Biochim Biophys Acta |Komary 2010]]
|| 
|| <div align="right">1</div>
|| 
|| 
|| <div align="right">2.5</div>
|| 
|| 

|}


== Amplex UltraRed in O2k-FluoRespirometry ==
===  [[O2k-Fluo Smart-Module]] and  [[O2k-Fluo LED2-Module]]===
:::: In high-resolution respirometry [[HRR]] the Amplex Red method<ref>[[Mohanty 1997 J Immunol Methods]]</ref> is used with the [[O2k-FluoRespirometer]] (O2k-Series D-G: [[O2k-Fluo LED2-Module]]) selecting the [[Fluorescence-Sensor Green]] and [[Smart Fluo-Sensor]] for [[O2k-FluoRespirometer#O2k-Series_H:_innovations |O2k-Series H]]. Use [[Stopper\black PEEK\conical Shaft\central Port |black stoppers]] with [[Cover-Slip\black |black cover-slips]] to exclude disturbances by external light sources. The fluorescence sensors are inserted though the windows of the O2k-Chambers<ref>[[Hickey 2012 J Comp Physiol B]]</ref>. The O2k has also been coupled to a fluorescence spectrophotometer with a light guide inserted through the [[Stopper\black PEEK\conical Shaft\central+side 2.5 mm Port|black PEEK stopper]].<ref> [[Anderson 2011 Am J Physiol Heart Circ Physiol]]</ref>

:::: The use of AmR in HRR to simultaneously determine respiration and H<sub>2</sub>O<sub>2</sub> flux has been intensively tested in the Oroboros lab (see references).

::::* AmR reacts with H<sub>2</sub>O<sub>2</sub> catalysed by [[Horseradish peroxidase]].

== 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 [http://tools.lifetechnologies.com/content/sfs/manuals/mp36006.pdf Manual Amplex UltraRed by Life Technologies].

::::# Dissolve one commercial vial of Ampler UltraRed (~1 mg) in 340 µL DMSO.
::::# Divide into 20 µL portions into 0.2 mL Eppendorf tubes.
::::# Store frozen at -20 °C in the dessicator.

::: '''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 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 H<sub>2</sub>O<sub>2</sub> production in an experimental run, including calibrations and chemical background conversion of AmR <ref> 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. - [[Makrecka-Kuka 2015 Biomolecules |»Bioblast link«]]</ref>,<ref> 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. - [[Komlodi 2018 Methods Mol Biol |»Bioblast link«]]</ref>.
:::: 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 H<sub>2</sub>O<sub>2</sub> production during the experiment (check by initial and final H<sub>2</sub>O<sub>2</sub> 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.

=== Horseradish peroxidase ===
:::: Titration into into 2 mL O2k-chamber: '''4 µL of HRP stock solution''', final concentration '''1 U/mL'''.

:::: [[Horseradish_peroxidase|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 H<sub>2</sub>O<sub>2</sub> from superoxide.

:::: (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.
:::: Experiments can be performed in the absence of SOD.     

== Calibration with H<sub>2</sub>O<sub>2</sub> ==

:::: Calibration standards of H<sub>2</sub>O<sub>2</sub>: Commercial solution (Sigma-Aldrich 323381 - 25 mL  hydrogen peroxide solution 3 wt.%; stabilized with acetanilide, c. 200 ppm) = 880 mM H<sub>2</sub>O<sub>2</sub>. 
::::# Prepare a HCl stock solution of 10 µM HCl.
::::# H<sub>2</sub>O<sub>2</sub> dilution 1 (1:88): add 10 µL of a commercial stock solution of 3 wt.  H<sub>2</sub>O<sub>2</sub> to 870 µL 10 µM HCl solution, to obtain a 10 mM  H<sub>2</sub>O<sub>2</sub> solution.
::::# H<sub>2</sub>O<sub>2</sub> dilution 2 (1:250):add 4 µL of 10 mM  H<sub>2</sub>O<sub>2</sub> solution to 996 µL 10 µM HCl solution, to obtain the  H<sub>2</sub>O<sub>2</sub> stock solution of 40 µM.
:::: The H<sub>2</sub>O<sub>2</sub> calibration solution has to be prepared fresh every day, kept away from light exposure, and avoid freezing it.

::::* Titration into into 2 mL O2k-Chamber: '''5 µL of the 40 µM H<sub>2</sub>O<sub>2</sub> stock''', step increase of '''0.1 µM  H<sub>2</sub>O<sub>2</sub>'''.

:::::'''Problem:''' ''Here is a DatLab file where I ran the H2O2 calibration protocol with 6 H<sub>2</sub>O<sub>2</sub> titrations.  I drew up 5 uL of H<sub>2</sub>O<sub>2</sub> into the syringe for titration into each chamber individually for the first two titrations.  For the remaining titrations, I drew up 10 uL of H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> was added to the chambers, it can be seen that the chamber which is titrated second when 10 uL of H<sub>2</sub>O<sub>2</sub> is drawn into the syringe at one time has reduced sensitivity measurements.  When fresh H<sub>2</sub>O<sub>2</sub> 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.)
:::::[[File:Jennifer Norman 1.png|400px]] [[File:Jennifer Norman 2.png|400px]]

:::: '''Answer:''' It is recommended to fill the syringe with H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> titration is not precise filling it up with the maximal volume or H<sub>2</sub>O<sub>2</sub> might be degraded by light already in the syringe.

::::* Background calibration- quality control: One to three subsequent H<sub>2</sub>O<sub>2</sub> titrations after addition of respiration medium, HRP and Amplex UltraRed to the O2k-Chamber but before sample addition. This step is required for the calculation of the theoretical chemical background fluorescence (see [[MiPNet24.10 H2O2 flux analysis]] and https://bioblast.at/index.php/Flux_/_Slope#How_to_Analyse_with_DatLab_7.4).
::::* 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 H<sub>2</sub>O<sub>2</sub>. 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 H<sub>2</sub>O<sub>2</sub> calibrations when using the Amplex UltraRed (AmR) assay?'''
:::::The sensitivity to H<sub>2</sub>O<sub>2</sub> of the Amplex UltraRed assay changes during the experiment, thus requires multiple calibrations. These sensitivity changes may be the function of 1) experimental time, 2) changes in the optical properties of the respiration medium due to sample/chemical titration, 3) the radical scavenging capacity of the sample, and 4) the concentration of accumulating resorufin (UltroxRed in the case of Amplex UltraRed). Therefore, it is recommended to perform H<sub>2</sub>O<sub>2</sub> calibration before sample addition, after sample addition and multiple times during the experiment.- »[[Komlodi 2018 Methods Mol Biol]]« 
:::: The DLPs for AmR SUIT protocols already come with multiple steps of H<sub>2</sub>O<sub>2</sub> calibrations implemented.- » [[SUITbrowser]]« 
::::* [[Amp calibration - DatLab|DatLab]] supports automatic H<sub>2</sub>O<sub>2</sub> calibration by calculating the calibration parameters by linear regression and graphical display of the calibration regression.




=== Experimental media for the AmR assay ===

:::: Media with high antioxidant activity compete with HRP and partially consume H<sub>2</sub>O<sub>2</sub> before it can react with AmR to form the active fluorophore UltroxRed (similar to resorufin). This was shown by comparing the resorufin-sensitivity and H<sub>2</sub>O<sub>2</sub>-sensitivity <ref> 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. - [[Krumschnabel 2015 Methods Mol Biol |»Bioblast link«]]</ref>,<ref> 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. - [[Komlodi 2018 Methods Mol Biol |»Bioblast link«]]</ref>.
::::* H<sub>2</sub>O<sub>2</sub>-sensitivity: the change of fluorescence signal per µM H<sub>2</sub>O<sub>2</sub> added in calibrations with H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub>-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.
=== Calculation of theoretical fluorescence background slope of MiR05 ===
::::* in progress
=== HRP-independent background AmR flux ===

:::: 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 H<sub>2</sub>O<sub>2</sub> 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.


:::: A study by Miwa et al 2016 (Free Radical Bio Med 90:173) suggests that this phenomenon 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 [http://bioblast.at/index.php/Talk:Amplex_red here].

:::: For further informaton see the Discussion page.

== 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, H<sub>2</sub>O<sub>2</sub>) can cause artefacts as observed in the figure: 
:::: [[File:Injection artefact AmR.png|900px]]

:::: The marks should always be set on the stable signal avoiding the artefacts area.
:::: [[File:Injection artefact AmR marks.png|900px]]
=== NADH and reduced glutathione ===
:::: Votyakova TV and Reynolds IJ (Archives of Biochemistry and Biophysics 431(1):138-44. 2014) revealed that NADH and the reduced glutathione is able to react with AmR in the presence of O<sub>2</sub> 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

::::* Inhibitors of [[horseradish peroxidase]], e.g. cyanide, azide; more information: [[horseradish peroxidase]]

::::* Catalase and other substances consuming or scavenging H<sub>2</sub>O<sub>2</sub>. The effect of substances in the medium that consumes H<sub>2</sub>O<sub>2</sub> slowly 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:''[https://wiki.oroboros.at/index.php/Amplex_UltraRed#Avoiding_artefacts| 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 H<sub>2</sub>O<sub>2</sub>), [[succinate]],  ([[ADP]] + Mg<sup>2+</sup>), ([[ATP]] +  Mg<sup>2+</sup>), [[rotenone]], [[FCCP]], [[CCCP]], [[oligomycin]],  [[antimycin A]], [[malonate]], [[myxothiazol]]

=== Avoiding artefacts ===

:::: The  Amplex method is based on the H<sub>2</sub>O<sub>2</sub> dependent oxidation of AmR to UltroxRed by HRP. Under unfavorable conditions AmR  may be oxidized even in the absence of  H<sub>2</sub>O<sub>2</sub>. 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 H<sub>2</sub>O<sub>2</sub> 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  H<sub>2</sub>O<sub>2</sub> production. If a  significant Amp slope is detected before the addition of HRP this increase in fluorescence is not caused by  H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub>. Then the apparent H<sub>2</sub>O<sub>2</sub> flux (the Amp slope) should be reduced to near zero as a control for distinguishing an unspecific chemical background slope from H<sub>2</sub>O<sub>2</sub> 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 ==
::::* To decrease the oxygen concentration using nitrogen see [[Setting the oxygen concentration]].

== Experimental SOP==
::::* [[MiPNet20.14 AmplexRed H2O2-production]]
::::* [[MiPNet18.05 Amplex-Mouse-heart]]
::::* [[MiPNet18.06 Amplex-Yeast]]
== Permeabilized fibres==
:: '''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:''[https://wiki.oroboros.at/index.php/Permeabilized_muscle_fibers#Oxygen_dependence_of_ROS_production_-_are_permeabilized_fibers_a_valid_model.3F Oxygen dependence of pfi]
== H<sub>2</sub>O<sub>2</sub> flux analysis and mark setting ==

::::* '''Mark setting:''' The black line refers to the raw fluorescence signal [V] of the product of the AmR assay called resorufin (UltroxRed in the case of Amplex UltraRed), while the green line is the fluorescence slope [mV/s] automatically calculated from the black line.

::::* Excel templates are provided for analysis of the H<sub>2</sub>O<sub>2</sub> 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).

::::* [[MiPNet24.10 H2O2 flux analysis ]]
::::* The calculations used in the excel analysis template are provided complying with Oroboros transparency policy: [https://wiki.oroboros.at/index.php/Flux_/_Slope#H2O2]
::::* [https://wiki.oroboros.at/index.php/Smoothing#DatLab_Amp_slope:_performance_and_data_analysis| Amp slope smoothing in DatLab]


{{Keywords: H2O2}}
== 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 H<sub>2</sub>O<sub>2</sub> flux? ===
:::*''-See:''
::::[[MiPNet20.14 AmplexRed H2O2-production]]
::::[[Smoothing]]
=== How to analyse the data in the Excel template? ===
:::*''-See:''
::::[[MiPNet24.10 H2O2 flux analysis]]
::::[https://wiki.oroboros.at/index.php/Flux_/_Slope#How_to_Analyse_with_DatLab_7.4| H2O2 analysis]

== References ==
<references/>
::::* [[Komary 2010 Biochim Biophys Acta]]
::::* [[Tretter 2012 Free Radic Biol Med]]
::: '''O2k-Demo experiments'''
::::* [[MiPNet20.14 AmplexRed H2O2-production]]
::::* [[MiPNet18.05 Amplex-Mouse-heart]]
::::* [[MiPNet18.06 Amplex-Yeast]]
::::* [[MiPNet24.10 H2O2 flux analysis ]]
{{MitoPedia concepts}}
{{MitoPedia methods
|mitopedia method=Fluorometry
}}
{{MitoPedia O2k and high-resolution respirometry}}
{{MitoPedia topics}}