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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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| {| style="border-spacing:0;"
| | :::: [[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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| |-
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| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| Amplex Red
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| | 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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| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| Amplex Ultra Red
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| | 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">48.8</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">A36006</div>
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| |-
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| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| Ampliflu Red
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| | style="border:none;padding-top:0cm;padding-bottom:0cm;padding-left:0.053cm;padding-right:0.053cm;"| <center>Sigma</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">18.5</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">90101</div>
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| |-
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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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| {| border="1" style="border-spacing:0;" style="text-align: center;"
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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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| |-
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| || stock
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| || final
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| || unit definition
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| || stock
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| || final
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| |-
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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_Anal_Biochem|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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| |-
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| || [[Zhou_1997_Anal_Biochem|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)</div>
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| || [[Mohanty_1997_J_Immunol_Methods|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 used two publications from [[HU Budapest Tretter L|Prof. Tretters OROBOROS MiPNet Reference Laboratory]] as a starting point for our development: [[Tretter 2012 Free Radic Biol Med]] and [[Komary 2010 Biochim Biophys Acta]].
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| == Media ==
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| Media that were designed to have some antioxidant properties will frequently consume H2O2 before it can react with Amplex (R) (Ultra)Red to form the active fluorophore resorufin. This can be shown by comparing
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| * the sensitivity as determined by H2O2 additions to different media containing HRP and Amplex (R) UltraRed
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| * the sensitivity as determined by addition of the actual fluorophore resorufin to the same media (signal change per added resorufin)
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| (Sensitivity is signal change per added H2O2 or resorufin.)
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| While the sensitivity measured by the addition of H2O2 drops drastically from a simple phosphate buffer to media with strong antioxidant properties, this is not observed when the sensitivity is determined by the addition of resorufin.
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| Since the non biological drift always observed for the Amplex /HRP system is independent of the sensitivity versus H2O2 the same (absolute) drift expressed as (calibrated) "apparent H2O2 production" will be higher in a medium with low H2O2 sensitivity as compared to a medium with high H2O2 sensitivity.
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| As expected some traditional KCl based media, similar to those used in ... showed a far higher sensitivity against H2O2 addition than MiR05, while some experiments indicated a lower respiration.
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| === Comparison MiR05Cr to MiRK03Cr ===
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| '''Conditions:'''
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| ''Instrumental settings:''
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| *Amp.Gain 1000
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| *Light intesity variable 1mA, 2mA and 5mA
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| *Configuration O2K -Sensor always the same: Oxygraph AB: Sensors B-0111/B-0082 Oxygraph CD: Sensors B-0119/B-0117 Oxygraph EF: Sensors A-0004/B-0112 Oxygraph GH: Sensors A-0087/A-0088
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| ''Media:''
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| [[MiR05Cr]] and
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| MiRK03Cr:
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| KCl 130 mM,
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| HEPES free acid 20 mM,
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| KH2PO4 10 mM,
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| MgCl2 3 mM,
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| EGTA 0.5 mM,
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| BSA : 0.1%,
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| pH=7,
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| add 120 mg Creatine to 40 ml buffer
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| '''Titration:'''
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| 10 µl Amplex UltraRed (conc:1 mM -> 5 µMl in chamber)
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| 4µl HRP (conc:500 U/ml -> 1 U/ml in chamber)
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| 2*10 µl of 56,32 µM H2O2 solution
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| '''Results/Figures:'''
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| '''Drift vs Sensitivity:'''
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| [[Image:Drift vs sensitivity.png|600px]]
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| Fig.6
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| [[image:SD drift at diff. H2O2 conc.png|600px]]
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| Fig.7
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| --> Drift and sensitivity at least as good as MitoOx2 (=MiRK02)
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| ''' Drift vs light intensity'''
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| [[Image:Light int. vs. calib.drift in MiR05Cr.png|600px]]
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| [[Image:Light int. vs.calib. drift in MiRK03Cr.png|600px]]
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| Fig.14 and Fig. 15
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| --> MirK03, MiR05: drift not dependent on light intensity ( 1 to 5 mA)
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| compare to "MitoOx2":
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| [[Image:Drift vs light MitoOx2.png|600px|left]]
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| '''Evaluation of respirometry in MiR05Cr versus MiRK03Cr (N=2):'''
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| '''Titration:'''
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| 10 µl Amplex UltraRed (conc:1 mM -> 5 µM in chamber)
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| 4µl HRP (conc:500 U/ml -> 1 U/ml in chamber)
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| 2*5 µl of 28.16 µM H2O2 solution : 0 µM, 0.07040 µM, 0.1406 µM
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| [[File:O2_Flux_per_mass_MiR05Cr_vs_MiRK03Cr.png]]
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| [[File:Flux_Control_Ratio_MiR05Cr_vs_MiRK03Cr.png]]
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| Fig.16 and 17
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| To do:? with switched chambers with and without Creatine in low light intensity
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| [[File:Drift signal ratios.png]]
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| Fontanam 16:22, 6 August 2013 (CEST)
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| | == Additional references == |
| | ::::* [[Perevoshchikova 2013 Free Radic Biol Med]] |
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