Difference between revisions of "Talk:Amplex UltraRed"

Latest revision as of 22:03, 3 August 2023

MiPNet discussion forum: HRP independent background flux (2016-07-25)

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.

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.

Purhonen J

Additional references

Perevoshchikova 2013 Free Radic Biol Med

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-__TOC__
+== MiPNet discussion forum: HRP independent background flux (2016-07-25) ==
+=== 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.
-=== Comparison MiR05Cr to MiRK03Cr ===
+:::: 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.
-'''Instrumental settings'''
+:::: [[Purhonen J]]
-* Amp. Gain 1000
-* Light intesity variable: 1 mA, 2 mA and 5 mA
-* Configuration O2k-Fluo sensor always the same: O2k P1: Sensors B-0111/B-0082; O2k P2: Sensors B-0119/B-0117; O2k P3: Sensors A-0004/B-0112; O2k P4: Sensors A-0087/A-0088.
-'''Media'''
-* [[MiR05Cr]]
-* MiRK03Cr: KCl 130 mM, HEPES free acid 20 mM, KH2PO4 10 mM, MgCl2 3 mM, EGTA 0.5 mM, BSA 0.1%, pH=7, add 120 mg Creatine to 40 ml buffer. After thawing MiRK03 a preciptate was observed, which was dissolved after 20 min shaking.
-'''Titrations'''
-* 10 µl Amplex UltraRed (stock: 1 mM -> 5 µM final in chamber)
-* 4 µl HRP (stock: 500 U/ml -> 1 U/ml final in chamber)
-* 2*10 µl of 56,32 µM H<sub>2</sub>O<sub>2</sub> solution
-'''Results/Figures'''
-'''Drift vs Sensitivity:'''
+== Additional references ==
-* See [[Krumschnabel 2015 Methods Mol Biol]].
+::::* [[Perevoshchikova 2013 Free Radic Biol Med]]
-[[Image:Drift vs sensitivity.png|600px]]
-Fig.6
+[[Image:BB-Bioblast.jpg|left|30px|link=http://www.bioblast.at/index.php/Bioblast:About|Bioblast wiki]]
-[[image:SD drift at diff. H2O2 conc.png|600px]]
-Fig.7
---> Drift and sensitivity at least as good as MitoOx2 (=MiRK02)
-''' Drift vs light intensity'''
-[[Image:Light int. vs. calib.drift in MiR05Cr.png|600px]]
-[[Image:Light int. vs.calib. drift in MiRK03Cr.png|600px]]
-Fig.14 and Fig. 15
---> MirK03, MiR05: drift not dependent on light intensity ( 1 to 5 mA)
-compare to "MitoOx2":
-[[Image:Drift vs light MitoOx2.png|600px|left]]
-'''Evaluation of  respirometry in MiR05Cr versus MiRK03Cr (N=2):'''
-'''Titrations'''
-* 10 µl Amplex UltraRed (stock: 1 mM -> 5 µM final in chamber)
-* 4 µl HRP (stock: 500 U/ml -> 1 U/ml final in chamber)
-* 2*5 µl of 28.16 µM H<sub>2</sub>O<sub>2</sub> solution : 0 µM, 0.07040 µM, 0.1406 µM
-[[File:O2_Flux_per_mass_MiR05Cr_vs_MiRK03Cr.png]]
-[[File:Flux_Control_Ratio_MiR05Cr_vs_MiRK03Cr.png‎]]
-Fig.16 and 17
-[[File:Drift signal ratios.png]]
-Fontanam 16:22, 6 August 2013 (CEST)
-[[Image:BB-Bioblast.jpg|left|30px|link=Bioblast:About|Bioblast wiki]]
 == Popular Bioblast page ==
-[[Amplex red]] has been accessed more than
+::: [[Amplex UltraRed]] has been accessed more than
-:* 25,000 times (2015-10-29)
+::::* 50,000 times (2019-12-11)
-:* 20,000 times (2015-08-11)
+::::* 45,000 times (2019-07-22)
-:* 10,000 times (2015-04-27)
+::::* 40,000 times (2018-10-18)
-:*  5,000 times (2014-05-29)
+::::* 35,000 times (2016-10-12)
+::::* 30,000 times (2016-03-02)
+::::* 25,000 times (2015-10-29)
+::::* 20,000 times (2015-08-11)
+::::* 10,000 times (2015-04-27)
+::::*  5,000 times (2014-05-29)