Amplex red

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Amplex red

Description

Amplex red (AmR) is used as an extrinsic fluorophore for measurement of hydrogen peroxide production (ROS) by cells or mitochondrial preparations. The reaction of H2O2 and AmR is catalyzed by horseradish peroxidase to produce the red fluorescent compound resorufin (excitation wavelength 563 nm, emission 587 nm). The change of emitted fluorescence intensity is directly proportional to the concentration of H2O2 added, whereby the H2O2 is consumed.

Abbreviation: AmR

Reference: Mohanty 1997 J Immunol Methods, Zhou 1997 Anal Biochem, Mishin 2010 Free Radical Biol Med, Towne 2004 Anal Biochem, Krumschnabel 2015 Methods Mol Biol



MitoPedia methods: Fluorometry 




Contents


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O2k-technical support

This information is part of O2k-technical support and open innovation.

Sources of AmR

Trade Mark Manufacturer/ Distributor
price [€/mg]
product id
Amplex Red
Life Technologies
37.4
A12222
Amplex Ultra Red
Life Technologies
48.8
A36006
Ampliflu Red
Sigma
18.5
90101
Quanta Red
Thermo scientific
15150


Citation Amplex HRP pH Limit of detection
stock final unit definition stock final
mM
µM
U/ml
U/ml
BIOTEK
10
50
pyrogallol
10
0.1
7.4
4 nM (absorption 300 nm)
Life Technologies
10
50
10
0.1
6 to 7.5
<80 nM
Towne 2004
160
0.41
7.5 to 8.5
100 nM
Zhou1997
3 ?
0.3 to 1
50 nM (10 nM optimal)
Mohanty 1997
100
10 to 100
1
18 nM
Komary 2010
1
2.5


Amplex red in O2k-Fluorometry

The use of AmR in HRR to simultaneously determine respiration and H2O2 flux has been intensively tested in the OROBOROS lab (see references).
  • Source: Life Technologies (former Invitrogen) A36006.
  • AmR, similar to other chemical probes, exerts a certain degree of toxicity and should thus be used at the lowest concentration compatble with the total H2O2 production in an experimental run, including calibrations and chemical background conversion of AmR (Makrecka-Kuka et al 2015).
  • In the absence of sample, there is a spontaneous increase of the resorufin fluorescence signal over time, the extent of which depends on components of the respiration medium (Krumschnabel et al 2015). The sensitivity – the change in fluorescence per unit of H2O2 produced – depends on the medium 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 in data analysis, for which DatLab-Analysis templates are available.
  • Media optimized for fluorometry with AmR are not necessarily 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 fluorometry.


Antioxidant capacity of experimental media

Media with high antioxidant activity compete with HRP and partially consume H2O2 before it can react with AmR to form the active fluorophore resorufin. This was shown by comparing the resorufin-sensitivitiy and H2O2-sensitivity (Krumschnabel et al 2015).
  • H2O2-sensitivity: the change of fluorescence signal per µM H2O2 added in calibrations with H2O2 titration in media containing HRP and AmR.
  • Resorufin-sensitivity: the change of fluorescence signal per µM resorufin added in calibrations with resorufin titration in media containing HRP and AmR.
The H2O2-sensitivity is much higher in a simple phosphate buffer compared to media with strong antioxidant capacity. In contrast, this is not the case for the resorufin-sensitivity.


HRP independent background AmR flux

In some experiemental systems an increase of resorufin fluorescence was observed that appeared independent of HRP activity. A study by Miwa et al. (Free Radical Bio Med 2016; 90:173) suggests that this phenomenon is related to a mitochondrially expressed carboxylesterase which converts Amplex Red to resorufin at a high rate. The issue can be relatively easily solved by adding a protease inhibitor, an approach that seems to work in various mitochondrial preparations as described here.


References

  • Komary 2010 Biochim Biophys Acta
  • Tretter 2012 Free Radic Biol Med
  • Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. »Bioblast pdf«
  • Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38. »Bioblast pdf«
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