Talk:Amplex UltraRed

• Tretter 2012 Free Radic Biol Med
• Komary 2010 Biochim Biophys Acta.

Media

See Krumschnabel 2015 Methods Mol Biol.

Media with high antioxidant activity compete with HRP and partially consume H₂O₂ before it can react with Amplex (R) (Ultra)Red to form the active fluorophore resorufin. This was shown by comparing

• the sensitivity as determined by H₂O₂ additions to different media containing HRP and Amplex (R) UltraRed;
• the sensitivity as determined by addition of the actual fluorophore resorufin to the same media (signal change per added resorufin); the sensitivity is signal change per added H₂O₂ or resorufin.

The sensitivity, as determined by addition of H₂O₂, is much higher in a simple phosphate buffer compared to media with strong antioxidant capacity. In contrast, this is not the case for the sensitivity with respect to resorufin.

A chemical background drift, which is always observed for the Amplex /HRP system, is independent of the H₂O₂ sensitivity versus 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.

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.

Comparison MiR05Cr to MiRK03Cr

Instrumental settings

• 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 H₂O₂ solution

Results/Figures

Drift vs Sensitivity:

• See Krumschnabel 2015 Methods Mol Biol.

Fig.6

Fig.7

--> Drift and sensitivity at least as good as MitoOx2 (=MiRK02)

Drift vs light intensity

Fig.14 and Fig. 15

--> MirK03, MiR05: drift not dependent on light intensity ( 1 to 5 mA)

compare to "MitoOx2":

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 H₂O₂ solution : 0 µM, 0.07040 µM, 0.1406 µM

Fig.16 and 17

Fontanam 16:22, 6 August 2013 (CEST)

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