Amplex UltraRed

high-resolution terminology - matching measurements at high-resolution

Amplex UltraRed

Description

Amplex red (AmR) belongs to the extrinsic fluorophores and makes use of the fact that ROS generate hydrogen peroxide (H₂O₂) and it is the H₂O₂ level that Amplex red is used to measure. In the presence of horseradish peroxidase, Amplex red reacts with H₂O₂ to produce the red fluorescent compound resorufin (excitation wavelength 563 nm, emission 587 nm). The emitted fluorescence intensity is directly proportional to the H₂O₂ concentration.

Abbreviation: AmR

Reference: Mishin 2010 Free Radical Biol Med, Towne 2004 Anal Biochem, Hickey 2012 J Comp Physiol B, Fasching 2011 Abstract Berlin

MitoPedia methods: Fluorometry 

The Amplex Red method can be used with the O2k-Fluorescence LED2-Module. In addition, the Oxygraph-2k has been coupled to full fluorescence spectrophotometers for this purpose, with a light guide inserted through the black PEEK stopper.

Preparation of Amplex® UltraRed Solutions

Source: Invitrogen A36006

Storage solution

Prepare a 10 mM storage solution of Amplex® UltraRed reagent (AmR) by adding 340 μL of fresh, high‑quality DMSO (Sigma D8418) to one commercial vial of Amplex® UltraRed reagent (1 mg). Vortex well to dissolve. Protect AmR from light and moisture. Store commercial AmR and storage solutions in the dark, protected from moisture at –20°C for future use. When stored properly, the 10 mM storage solution is stable for at least 6 months.

Note: The preparation of the 10 mM storage solution follows the procedure suggested by the manufacturer. The manufacturer states only an approximate molecular weight for the Amplex® UltraRed formulation and does not publish details how the Amplex® UltraRed formulation deviates from the substance 10-acetyl-3,7-dihydroxyphenoxazine (CAS# 119171-73-2), known as Amplex ® Red.

Stock solution

Dilute the 10 mM storage solution 1:10 with DMSO to get the 1 mM stock solution. Small aliquots (100 µl) of the 1 mM stock solution may be stored at -20°C.

Final concentration

Titrate 10 µl of 1 mM stock solution into the 2 ml O2k-chamber with MiR05 (or MiR05Cr, to obtain a final concentration of 5 µM AmR. Optimize the final AmR concentration according to respiration media, sample type and concentration and experimental protocol. These determine the total AmR consumption by H2O2 production during the experiment (check by initial and final H2O2 titrations for calibration). The final concentration of AmR becomes diminished during an experiment due to AmR consumption and dilution by titrations in a SUIT protocol.

Horse radish Peroxidase

Horse radish peroxidase (Sigma-Aldrich P 8250 - 5 kU): Stock solution 500 U HRP/ml MiR05 or MiR05Cr; can be used as storage solution at -20 °C. Titrate 4 µl of stock solution into the 2 ml O2k-chamber. Final concentration in chamber 1 U/ml.

Calibration with H₂O₂

• Add sample, HRP , and Amplex UltraRed to the O2k chamber
• Commercial solution (Sigma-Aldrich 323381 - 25 ml hydrogen peroxide solution 3 wt. %) = 880 mM.

Prepare fresh:

• Dilution 1 (1:125; solvent H2O): 200 µl commercial solution in 25 ml (total volume) = 7.04 mM.
• Dilution 2 (1:125; solvent H2O): 400 µl dilution 1 + 500 µl 1 mM HCl (for stabilization) in 50 ml = 56.32 µM stock solution.
• Titrate 2 * 10 µl of 56.32 µM stock solution into the 2 ml O2k-chamber = 2* 0.2816 µM H₂O₂ = total 0.5632 µM H₂O₂. If H₂O₂ concentrations are corrected for displacement of medium by subsequent injections the following nominal H₂O₂ concentrations in the chamber can be used for calibration purposes: 0 µM, 0.2816 µM, 0.5618 µM. We suggest to perform a calibration at the beginning (sample already present) and (for quality control) also near the end of an experiment (but before any substances are added that are incompatible with the method).

An spreadsheet template for the numerical evaluation of the H2O2 calibration is available for download @OROBOROS. Further instructions are found in the template under 'Help'.

Substances incompatible with the Amplex Red method

The following substances/ classes of substances are strictly incompatible with the Amplex Red method for theoretical reasons:

• strongly redox active substances, e.g. cytochrome c, TMPD/Ascorbate

• inhibitors of horseradish peroxidase, see horseradish peroxidase

• catalase and other substances consuming H₂O₂. The effect of substances in the medium that consume H₂O₂ slowly is taken account of by the calibration procedure. However, such substances will decreases the sensitivity of the method. Note that catalase can be a valuable tool for Amplex_red#Checking_for_artifacts Checking for artifacts.

The effect of other substances should be checked by blank experiments, including comparing the sensitivity (result of calibration) before and after injecting the substances.

In preliminary experiments Oroboros Instruments got indications that small amounts (as typically used in SUIT protocols) of the following substances are compatible with the method:

DMSO, ethanol, malate, glutamate, pyruvate, succinate, (ADP + Mg²⁺), (ATP + Mg²⁺), rotenone, FCCP, oligomycin, antimycin A, malonate, myxothiazol

Checking for artifacts

The Amplex method is based on the H₂O₂ dependent oxidation of Amplex Red to resorufin by HRP. Under unfavorable conditions Amplex Red may be oxidized even in the absence of H₂O₂. At a small rate such a oxidation occurs in the presence of HRP even without any sample present. The magnitude of this "Drift" depends inter alia on the used light intensity and can therefore be minimized by using the suggest or lower light intensity. Components of the sample may however induce a far higher, non H₂O₂ related rate of Ample Red oxidation. Therefore, especially when applying the method on new types of samples the method should be checked for artifacts. A few approaches are listed here:

Sequential addition of HRP and AmR: This method is particular easy to implement if Amplex Red and HRP have to be added to the chamber already containing the sample anyway: Inject first Amplex Red, wait a few minutes for flux stabilization. The Amp slope hast to stay near zero. Then add HRP. The Amp slope should increase and correspond to the H₂O₂ production. If a significant Amp slope is detected before the addition of HRP this increase in fluorescence is not caused by H₂O₂ production. The experiment can be continued as usual after this test. If the sample is injected routinely into the chamber already containing AmR and HRP the method can not be applied. In this case it is suggested to change this sequence at least for one experiment.

Addition of catalse: After a presumed H₂O₂ production rate is established a high dose (e.g. 10 µl of a 280000 u/ml stock solution) of catalase is injected. Because catalase competes with HRP for the available H₂O₂, the apparent H₂O₂ production rate (the Amp slope) has to be reduced nearly to zero. If the the Amp slope is unchanged or decreases only partially the increase in fluorescence is an artifact (non H₂O₂ dependent Amplex red oxidation). The experiment can NOT be continued afterwards!

References