Amplex UltraRed

high-resolution terminology - matching measurements at high-resolution

Amplex UltraRed

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

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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-Excel 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.

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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