View source for Amplex UltraRed

{{MitoPedia
|abbr=AmR
|description='''Amplex<sup>®</sup> 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 fluorescent product according to the supplier is called UltroxRed in the case of Amplex<sup>®</sup> UltraRed which has a 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=Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. https://doi.org/10.26124/bec:2021-0004
}}
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{{Technical support integrated}}

=== Amplex Red or Amplex UltraRed in research articles ===



{|  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® UltraRed method<ref>[[Mohanty 1997 J Immunol Methods]]</ref> 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-FluoRespirometer#O2k-Series_H:_innovations |O2k-Series H - I]]. 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 |references]]).

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

== Application 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].

::: '''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 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.

==== 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 H<sub>2</sub>O<sub>2</sub> 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'''.

:::: [[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 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 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 freezing should be avoided.

::::* Titration 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>'''.

::::* '''Background H<sub>2</sub>O<sub>2</sub> calibration:''' 
:::::* A set of H<sub>2</sub>O<sub>2</sub> 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 [[Flux_/_Slope#How_to_Analyse_with_DatLab_7.4| How to analyze H<sub>2</sub>O<sub>2</sub> flux in DatLab]], and (''2'') for calculation of the [[sensitivity]] of the Amplex® UltraRed assay towards H<sub>2</sub>O<sub>2</sub>, 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 H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub>, is prepared, or a different respiration medium is used. As the H<sub>2</sub>O<sub>2</sub> stock solution needs to be freshly prepared, it is recommended to perform a background H<sub>2</sub>O<sub>2</sub> calibration before each experiment and at a minimum once per experimental day.
:::::* The protocol for background H<sub>2</sub>O<sub>2</sub> 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 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 in 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 and accumulating UltroxRed (similar to resorufin), (''2'') changes of the optical properties due to titrations, (''3'') the radical scavenging capacity of the sample. Therefore, H<sub>2</sub>O<sub>2</sub> 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 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.

:::::'''Problem:''' ''Here is a DatLab file where I ran the H<sub>2</sub>O<sub>2</sub> 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 µL 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.


=== 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.

==== H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> equivalent concentration [µM]. If experiments present different sensitivities, the resulting H<sub>2</sub>O<sub>2</sub> 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 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.
::::* The Excel template is ideal for analysis of O<sub>2</sub> and H<sub>2</sub>O<sub>2</sub> 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:
:::::* The chemical background flux can be measured as follows: [[File:AmR background fluorescence slope.DLP]]
:::::*	In DatLab 7.4, set the marks to the Amp slope according to the protocol.
:::::: 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 [mV/s]´ 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). 
:::::* The Excel templates are available here: [[File:AmR background fluorescence slope.xlsx | AmR background fluorescence slope]] [[File:AmR background fluorescence slope demo.xlsx |AmR background fluorescence slope demo file]] 


=== 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 [http://bioblast.at/index.php/Talk:Amplex_red 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 H<sub>2</sub>O<sub>2</sub> 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, 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, 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 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 slowly consume H<sub>2</sub>O<sub>2</sub> 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 catalase:''' 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.

== Oxygen range ==
:::: The H<sub>2</sub>O<sub>2</sub> production of mitochondria is oxygen dependent [[Komlodi 2021 BEC AmR-O2]]. The oxygen concentration can be controlled in the O2k-chamber while running the AmR assay.  We recommend running the assay at low O<sub>2</sub> concentration, ''e.g.'', between 60 and 30 µM O<small>2</small>. If this is not possible, it is advisable to run all assays in the same [O<sub>2</sub>] range, and take marks for different conditions under a similar [O<sub>2</sub>] to minimize variability.

=== Nitrogen or hydrogen injection ===
::::* To decrease the oxygen concentration in the O2k-chamber using nitrogen or hydrogen see [[Setting the oxygen concentration]].
::::* Check [[Oxia]] for fast and safe production of hydrogen (and oxygen).

== 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:''[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 ==
::::* [https://wiki.oroboros.at/index.php/Smoothing#DatLab_Amp_slope:_performance_and_data_analysis| Amp slope smoothing in DatLab]
::::* [[MiPNet24.10 H2O2 flux analysis ]]
::::* [https://www.youtube.com/watch?v=_Fv0VSA_c14 H2O2 flux analysis video]
::::* '''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.

== Experimental SOP ==
::::* [[MiPNet20.14 AmplexRed H2O2-production]]
::::* [[MiPNet18.05 Amplex-Mouse-heart]]
::::* [[Komlodi 2021 BEC AmR-O2]]

== Excel analysis templates ==
::::* 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).
::::* The calculations used in the excel analysis template are provided complying with Oroboros transparency policy: [https://wiki.oroboros.at/index.php/Flux_/_Slope#H2O2]
::::* The excel analysis templates are updated for the respective SUIT protocol. The latest versions are available here (last update 2021-09-20):
:::::* For [[SUIT-018 AmR mt D041]] protocol, ''see'': [[File:SUIT-018 AmR mt D041 excel.xlsx]] and [[File:SUIT-018 AmR mt D041 demo.xlsx]]
:::::* For [[SUIT-026 AmR mt D064]] protocol, ''see'': [[File:SUIT-026 AmR mt D064 excel.xlsx]] and [[File:SUIT-026 AmR mt D064 demo.xlsx]]
::::* Manual: [[MiPNet24.10 H2O2 flux analysis ]]

== SUIT protocols ==
::::* [[SUIT-006 AmR mt D048]]: to study the influence of mt-membrane potential on NADH-linked H<sub>2</sub>O<sub>2</sub> flux
::::* [[SUIT-013 AmR ce D023]]: to detect H<sub>2</sub>O<sub>2</sub>2 flux in living cells
::::* [[SUIT-018]]: to detect NS-linked H<sub>2</sub>O<sub>2</sub> flux at different O<sub>2</sub> concentrations
::::* [[SUIT-026 AmR mt D064]]: to detect [[RET]]-linked H<sub>2</sub>O<sub>2</sub> flux

== Technical support ==

=== Set marks to calibrate the fluorescence signal using Amplex® UltraRed assay ===
::: To calibrate the fluorescence signal in molar units [µM], set the marks on the black plot.
::::» [[MiPNet20.14 AmplexRed H2O2-production]] 
::::» [[Amp calibration - DatLab]]

=== Set marks to analyse H<sub>2</sub>O<sub>2</sub> flux ===
::::» [[MiPNet20.14 AmplexRed H2O2-production]]
::::» [[Smoothing]]
::::» [https://www.youtube.com/watch?v=_Fv0VSA_c14 H2O2 flux analysis video]

=== Data analysis with the Excel template ===
::::» [[MiPNet24.10 H2O2 flux analysis]]
::::» [https://wiki.oroboros.at/index.php/Flux_/_Slope#How_to_Analyse_with_DatLab_7.4| H2O2 analysis]
::::» [https://www.youtube.com/watch?v=_Fv0VSA_c14 H2O2 flux analysis video]

=== Defective fluorescence module ===
:::*''-See:'' [[Defective fluorescence module]]

=== Exchange of filters in the fluorescence sensors ===
::::» [https://www.youtube.com/watch?v=eUukvgAi1yY Exchanging filter set of the Smart Fluo-Sensors]

=== How to connect the Smart Fluo-Sensors to the O2k? ===
::::» [https://www.youtube.com/watch?v=nlVLXcbBRqs Smart Fluo-Sensor assembly]

== References ==
<references/>
:* Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. https://doi.org/10.26124/bec:2021-0004
:* [[Komary 2010 Biochim Biophys Acta]]
:* [[Mishin 2010 Free Radical Biol Med]]
:* [[Towne 2004 Anal Biochem]]
:* [[Tretter 2012 Free Radic Biol Med]]
:* [[Zhou 1997 Anal Biochem]]

::: '''O2k-Demo experiments'''
::::* [[MiPNet20.14 AmplexRed H2O2-production]]
::::* [[MiPNet18.05 Amplex-Mouse-heart]]
::::* [[MiPNet24.10 H2O2 flux analysis ]]

{{Template:Keywords: H2O2}}

{{MitoPedia methods
|mitopedia method=Fluorometry
}}