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
Soources of AmR
Trade Mark | Manufacturer/ Distributor | price [€/mg]
|
product id
|
Amplex Red | 37.4
|
A12222
| |
Amplex Ultra Red | 48.8
|
A36006
| |
Ampliflu Red | 18.5
|
90101
| |
Quanta Red | 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
|
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.
- The H2O2-sensitivity is the change of fluorescence signal per µM H2O2 added in calibrations with H2O2 titration in different media containing HRP and AmR;
- the resorufin-sensitivity is the change of fluorescence signal per µM resorufin added in calibrations with resorufin titration in these media containing HRP and AmR.
- 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.
- 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.
Applications with O2k-Fluorometry
- Source: Life Technologies (former Invitrogen) A36006
- 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«
- Source: Life Technologies (former Invitrogen) A36006
- O2k-Demo experiments