Difference between revisions of "Talk:Amplex UltraRed"

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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:

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.

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.

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.

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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:
+::::* 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.
-=== Comparison MiR05Cr to MiRK03Cr ===
+::::* 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.
-'''Instrumental settings'''
+::::* 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.
-* 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:'''
-* See [[Krumschnabel 2015 Methods Mol Biol]].
-[[Image:Drift vs sensitivity.png|600px]]
-Fig.6
-[[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)