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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-The use of AmR in HRR to simultaneously determine respiration and H2O2 fluxes has been intensively tested in the OROBOROS lab and there are several points to be considered in its application:
+== MiPNet discussion forum: HRP independent background flux (2016-07-25) ==
-::::* AmR, similar to other chemical probes, exerts a certain degree of toxicity and should thus be used at the lowest concentration still allowing a proper detection of H2O2 production rates.
+=== 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.
-::::* Even in the absence of any sample, there is a spontaneous increase of the resorufin fluorescence signal over time detectable, the extent of which depends on components of the respiration medium used. Further, the assay sensitivity – the change in fluorescence per unit of H2O2 produced – is dependent on the medium used 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 when analyzing results of H2O2 production experiments.
+:::: 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.
-::::* Media that may minimize unwanted effects on fluorescence may not necessarily be optimal to support respiration and thus need to be carefully selected. At present, MiR05Cr and the new medium MiR07Cr (still under evaluation) appear to be the most suitable media for simultaneous respirometry and H2O2 flux detection.
+:::: [[Purhonen J]]
+== Additional references ==
+::::* [[Perevoshchikova 2013 Free Radic Biol Med]]
-[[Image:BB-Bioblast.jpg|left|30px|link=Bioblast:About|Bioblast wiki]]
+[[Image:BB-Bioblast.jpg|left|30px|link=http://www.bioblast.at/index.php/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)