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| |abbr=AmR | | |abbr=AmR |
| |description='''Amplex red''' (AmR) belongs to the [[extrinsic fluorophores]] and can be used as a probe to detect [[ROS]] production by cells or mitochondrial preparations in the form of [[hydrogen peroxide]]. 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 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. | | |description='''Amplex red''' (AmR) belongs to the [[extrinsic fluorophores]] and can be used as a probe to detect [[ROS]] production by cells or mitochondrial preparations in the form of [[hydrogen peroxide]]. 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 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]], [[Fasching 2011 Abstract Berlin]], [[Krumschnabel 2015 Methods Mol Biol]] | | |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]] |
| }} | | }} |
| {{MitoPedia methods | | {{MitoPedia methods |
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| {{MitoPedia topics}} | | {{MitoPedia topics}} |
| __TOC__
| | == Applications with O2k-Fluorometry == |
| == Application in HRR == | | :» '''O2k-Demo experiments''' |
| | | :* [[MiPNet18.06_Amplex-Yeast]] |
| The Amplex Red method<ref>[[Mohanty 1997 J Immunol Methods]]</ref> can be used with the [[O2k-Fluorescence LED2-Module]]<ref>[[Hickey 2012 J Comp Physiol B]]</ref> using the [[Fluorescence-Sensor Green]]. Use [[Stopper\black PEEK\conical Shaft\central Port |black stoppers]] with [[Cover-Slip\black |black cover-slips]] to exclude disturbances by external light sources.
| | :* [[MiPNet18.05_Amplex-Mouse-heart]] |
| | | :* [[MiPNet17.17_Amplex-Mouse-brain]] |
| The Oxygraph-2k has 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>
| | :* [[MiPNet19.20_AmplexRed_DataAcquisitionAnalysis]] |
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| For various application notes, please see: [[MiPNet18.06_Amplex-Yeast]], [[MiPNet18.05_Amplex-Mouse-heart]], [[MiPNet17.17_Amplex-Mouse-brain]].
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| === Preparation of Amplex® UltraRed solutions ===
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| Source: Life Technologies (former Invitrogen) A36006
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| See the manual provided by the producer: [http://tools.lifetechnologies.com/content/sfs/manuals/mp36006.pdf Manual Amplex UltraRed].
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| === Storage solution = stock solution ===
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| 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.
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| 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.
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| === Final AmR concentration ===
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| * Titration into into 2 ml O2k-chamber: '''2 µl of 10 mM AmR stock solution''', final concentration of '''10 µM AmR'''.
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| 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 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.
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| ==== Horse radish peroxidase ====
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| * Titration into into 2 ml O2k-chamber: '''4 µl of HRP stock solution''', final concentration '''1 U/ml'''.
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| [[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.
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| ==== Superoxide dismutase ====
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| [[Superoxide dismutase]] (SOD) is optionally included to generate H<sub>2</sub>O<sub>2</sub> 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.
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| (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.
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| === Calibration with H<sub>2</sub>O<sub>2</sub> ===
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| 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>.
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| # 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.
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| # H<sub>2</sub>O<sub>2</sub> dilution 1 (1:88): add 284 µl of a commercial solution of 3 wt.% H<sub>2</sub>O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> solution.
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| # H<sub>2</sub>O<sub>2</sub> dilution 2 (1:250): dilute 200 µl of 10 mM H<sub>2</sub>O<sub>2</sub> solution with 10 µM HCl to a volume of 50 ml to obtain the H<sub>2</sub>O<sub>2</sub> stock solution of 40 µM.
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| * 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>'''. One to three subsequent steps.
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| 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 H<sub>2</sub>O<sub>2</sub> measurement).
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| 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:
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| >> [[MiPNet19.18D_O2k-Calibration]].
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| Since part of the signal change might be caused by an ongoing H<sub>2</sub>O<sub>2</sub> production of the biological sample the calibration can be corrected by including the slope in the calculation.
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| Therefore
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| '''DatLab5 and DatLab 6'''
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| * Select the slope you want to include in the calculation (exclude slopes distorted by e.g. the injection of H2O2) by ticking the appropriate check boxes on the right side of the slope values. If you select more than one slope the mark duration weighted mean of all values is used for calculation.
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| * If for any reason the values of the slope displayed do not meet your expectations, e.g. too high value as an artifact due to the injection peak, instead of selecting you can set a mark on the Amplex slope manually and copy the corresponding value from 'Mark statistics' into the calibration window under "Independent slope". This slope will then be used for calculation.
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| '''DatLab 6:'''
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| In DatLab 6 the slope used for the correction is calculated from a linear regression signal vs time for the selected marks and take from the smoothed slope plot. This eliminates the distortion of the slope by injection artifacts after the Amp signal itself is already stable.
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| * Select the slope you want to include in the calculation by ticking the appropriate check boxes on the right side of the slope values. If you select more than one slope the weighted mean of all values is used for calculation.
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| all DatLab Versions:
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| * Finish your calibration with point 8. to 11. of the link mentioned above.
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| A spreadsheet template for the numerical evaluation of the H<sub>2</sub>O<sub>2</sub> calibration is available for download [http://www.oroboros.at/index.php?id=o2k-fluorescence @OROBOROS]. Further instructions are found in the template under 'Help'.
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| === Substances incompatible with the Amplex Red method ===
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| The following substances/ classes of substances are strictly incompatible with the Amplex Red method for theoretical reasons:
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| * Strongly redox active substances, e.g. cytochrome ''c'', TMPD/Ascorbate
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| * Inhibitors of [[horseradish peroxidase]], e.g. cyanide, azide; more information: [[horseradish peroxidase]]
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| * Catalase and other substances consuming or scavanging H<sub>2</sub>O<sub>2</sub>. The effect of substances in the medium that consume H<sub>2</sub>O<sub>2</sub> 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 [[Amplex_red#Checking_for_artifacts|checking for artifacts]]. | |
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| The effect of other substances should be checked by blank experiments, including comparing the sensitivity (result of calibration) before and after injecting the substances.
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| In preliminary experiments OROBOROS INSTRUMENTS evaluated small amounts (as typically used in SUIT protocols) of the following substances as '''compatible with the method''':
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| 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]], [[oligomycin]], [[antimycin A]], [[malonate]], [[myxothiazol]]
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| === Checking for artifacts ===
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| The Amplex method is based on the H<sub>2</sub>O<sub>2</sub> dependent oxidation of AmR to resorufin 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:
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| '''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.
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| '''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> production (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 Red oxidation.
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| Note: The experiment cannot be continued afterwards for measurement of hydrogen peroxide production, whereas respiration can be recorded onwards.
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| === Data acquisition and analysis ===
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| A step-by-step protocol of our current way of acquiring and analyzing AmR data is given in [[MiPNet19.20_Amplex_Red_Data_Acquisition_and_Analysis| MiPNet19.20]]
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| == References ==
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| <references/>
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