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
Media
See Krumschnabel 2015 Methods Mol Biol, Tretter 2012 Free Radic Biol Med, Komary 2010 Biochim Biophys Acta.
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.
Different brands
Trade Mark | Manufacturer/ Distributor | price [β¬/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
|
Β΅M
|
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
|
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. Β»Bioblast pdfΒ«
- 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. Β»Bioblast pdfΒ«
O2k-Demo experiments