Talk:Amplex UltraRed

Different Brands

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

Conditions

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)
INVITROGEN	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)
Mohanty1997	100	10 to 100			1		18 nM
Komary2010		1			2.5

At Oroboros Instruments we used two publications from Prof. Tretters OROBOROS MiPNet Reference Laboratory as a starting point for our development: Tretter 2012 Free Radic Biol Med and Komary 2010 Biochim Biophys Acta.

Media

Media that were designed to have some antioxidant properties will frequently consume H2O2 before it can react with Amplex (R) (Ultra)Red to form the active fluorophore resorufin. This can be 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)

(Sensitivity is signal change per added H2O2 or resorufin.)

While the sensitivity measured by the addition of H2O2 drops drastically from a simple phosphate buffer to media with strong antioxidant properties, this is not observed when the sensitivity is determined by the addition of resorufin.

Since the non biological drift always observed for the Amplex /HRP system is independent of the sensitivity versus H2O2 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 then MiR05, while some experiments indicated a lower respiration.

Media 5: Comparison MiR05Cr to MiRK03Cr

Conditions: Instrumental settings:

• Amp.Gain 1000
• Light intesity variable 1mA, 2mA and 5mA
• Configuration O2K -Sensor always the same: Oxygraph AB: Sensors B-0111/B-0082 Oxygraph CD: Sensors B-0119/B-0117 Oxygraph EF: Sensors A-0004/B-0112 Oxygraph GH: Sensors A-0087/A-0088

Media: MiR05Cr and 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

Titration:

10 µl Amplex UltraRed (conc:1 mM -> 5 µMl in chamber) 4µl HRP (conc:500 U/ml -> 1 U/ml in chamber) 2*10 µl of 56,32 µM H2O2 solution

Results/Figures:

Drift vs Sensitivity:

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":

Files: can be found in P:\3D_Mario\1_development_Fluorescence\amplex\MiRK03\DatLab data MiRK03Cr: 1 mA: 2013-04-30 AB02/CD02/EF02/GH02 2 mA: 2013-06-18 AB01/CD01/EF01/GH01 5 mA: 2013-06-18 AB02/CD02/EF02/GH02 MiR05Cr: 1mA: 2013-04-30 AB01/CD01/EF01/GH01 2mA: 2013-06-18 AB03/CD03/EF03/GH03 5mA: 2013-07-02 AB01/CD01/ 2013-06-28 EF01/GH01

Evaluation of respirometry in MiR05Cr versus MiRK03Cr (N=2):

Fig.16 and 17

Files to be found in P:\3C_Projects_Data\2013_all data\2013_Mouse Heart\Amplex red 2013-01-22 EF02.DLD (MiR05Cr) and 2013-01-22 GH02.DLD (MiRK03Cr) 2013-01-29 EF02.DLD (MiR05Cr) and 2013-01-29 GH02.DLD (MiRK03Cr) 2013-02-05 EF02.DLD (MiR05Cr) and 2013-02-05 GH02.DLD (MiRK03Cr)_ not included in Figure

To do:? with switched chambers with and without Creatine in low light intensity 

Fontanam 16:22, 6 August 2013 (CEST)