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| == Different Brands == | | == MiPNet discussion forum: HRP independent background flux (2016-07-25) == |
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| | === Janne Purhonen === |
| | :::: The problem was that I was observing a huge HRP independent sample related background. The assay was insensitive to addition of substrates and inhibitors. Then I came across a recent publication by Miwa et al. (Free Radical Bio Med 2016; 90:173 <http://www.sciencedirect.com/science/article/pii/S0891584915011090> –183). They nicely showed that Amplex Red can cross the mitochondrial membranes, but HRP cannot, and the liver mitochondria express carboxylesterase which converts the Amplex Red to resorufin at high rate. The good thing is that this carboxylesterase can be inhibited for example by a commonly used serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) at 100 µM which has marginal or no effect on mitochondrial respiration. |
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| | :::: My own experimentations showed that 100 µM PMSF reduced HRP independent sample related “H2O2” flux by 90% in the case of liver mitochondria and apparently completely eliminated this background in the case of kidney mitochondria. I also tested the PMSF in Amplex Red assay for isolated ''Drosophila melanogaster'' mitochondria in which the background is not that big issue as it is with the liver and kidney mitochondria. Anyway, PMSF eliminated the HRP-independent sample related background, thus inclusion of PMSF may improve the sensitivity of the assay not just with liver and kidney samples but with other sample types as well. |
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| | :::: [[Purhonen J]] |
| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| '''Trade Mark'''
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| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| '''Manufacturer/ Distributor'''
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| | 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>
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| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">'''product id'''</div>
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| | == Additional references == |
| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| Amplex Red
| | ::::* [[Perevoshchikova 2013 Free Radic Biol Med]] |
| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <center>Invitrogen</center>
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| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">37.4</div>
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| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">A12222</div>
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| | [[Image:BB-Bioblast.jpg|left|30px|link=http://www.bioblast.at/index.php/Bioblast:About|Bioblast wiki]] |
| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| Amplex Ultra Red | | == Popular Bioblast page == |
| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <center>Invitrogen</center> | | ::: [[Amplex UltraRed]] has been accessed more than |
| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">48.8</div>
| | ::::* 50,000 times (2019-12-11) |
| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">A36006</div>
| | ::::* 45,000 times (2019-07-22) |
| | | ::::* 40,000 times (2018-10-18) |
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| | ::::* 35,000 times (2016-10-12) |
| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| Ampliflu Red
| | ::::* 30,000 times (2016-03-02) |
| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <center>Sigma</center>
| | ::::* 25,000 times (2015-10-29) |
| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">18.5</div>
| | ::::* 20,000 times (2015-08-11) |
| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">90101</div>
| | ::::* 10,000 times (2015-04-27) |
| | | ::::* 5,000 times (2014-05-29) |
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| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| Quanta Red
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| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <center>Thermo scientific</center>
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| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"|
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| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <div align="right">15150</div>
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| |}
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| == Conditions ==
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| {| style="border-spacing:0;"
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| || Citation
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| | colspan="2" | Amplex
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| | colspan="3" | HRP
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| || pH
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| || Limit of detection
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| || <div align="right">stock</div>
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| || <div align="right">work</div>
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| || <div align="right">unit definition</div>
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| || <div align="right">stock</div>
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| || <div align="right">work</div>
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| || <div align="right">mM</div>
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| || <div align="right">µM</div>
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| || <div align="right">U/ml</div>
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| || <div align="right">U/ml</div>
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| || BIOTEK
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| || <div align="right">10</div>
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| || <div align="right">50</div>
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| || <div align="right">pyrogallol</div>
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| || <div align="right">10</div>
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| || <div align="right">0.1</div>
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| || <div align="right">7.4</div>
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| || <div align="right">4 nM (absorption 300 nm)</div>
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| || INVITROGEN
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| || <div align="right">10</div>
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| || <div align="right">50</div>
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| || <div align="right">10</div>
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| || <div align="right">0.1</div>
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| || <div align="right">6 to 7.5</div>
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| || <div align="right"><nowiki><80 nM</nowiki></div>
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| || Towne 2004
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| || <div align="right">160</div>
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| || <div align="right">0.41</div>
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| || <div align="right">7.5 to 8.5</div>
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| || <div align="right">100 nM</div>
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| || Zhou1997
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| || <div align="right">3 ?</div>
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| || <div align="right">0.3 to 1</div>
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| || <div align="right">50 nM (10 nm optimal cond)</div>
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| || Mohanty1997
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| || <div align="right">100</div>
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| || <div align="right">10 to 100</div>
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| || <div align="right">1</div>
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| || <div align="right">18 nM</div>
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| || [[Komary 2010 Biochim Biophys Acta|Komary2010]]
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| || <div align="right">1</div>
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| || <div align="right">2.5</div>
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| |}
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| At Oroboros Instruments we use the publication from [[HU Budapest Tretter L|Prof. Tretters OROBOROS MiPNet Reference Laboratory]] as a starting point for our development.
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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]], see [[horseradish peroxidase]]
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| * catalase and other substances consuming 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 will decreases the sensitivity of the method. Note that catalase can be a value tool for [[Talk: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 got indications that small amounts (as typically used in SUIT protocols) of the following substances are '''compatible''' with the method:
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| DMSO, ethanol, [[malate]], [[glutamate]], [[pyruvate]], [[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 Amplex Red to resorufin by HRP. Under unfavorable conditions Amplex Red may be oxidized even in the absence of H<sub>2</sub>O<sub>2</sub>. At a small rate such a oxidation occurs in the presence of HRP even without any sample present. The magnitude of this "Drift" depends inter alia on the used light intensity and can therefore be minimized by using the suggest 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 Ample Red 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 Amplex Red and HRP have to be added to the chamber already containing the sample anyway: Inject first Amplex Red, wait a few minutes for flux stabilization. The Amp slope hast 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 presumed H<sub>2</sub>O<sub>2</sub> production rate is established a high dose (e.g. 10 µl of a 280000 u/ml stock solution) of catalase is injected. Because catalase competes with HRP for the available H<sub>2</sub>O<sub>2</sub>, the apparent H<sub>2</sub>O<sub>2</sub> production rate (the Amp slope) has to be reduced nearly to zero. If the the Amp slope is unchanged or decreases only partially the increase in fluorescence is an artifact (non H<sub>2</sub>O<sub>2</sub> dependent Amplex red oxidation). The experiment can NOT be continued afterwards!
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