Amplex UltraRed

high-resolution terminology - matching measurements at high-resolution

Amplex UltraRed

Description

Amplex red (AmR) belongs to the extrinsic fluorophores and can be used as a probe to detect ROS production in mitochondria. It 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: Zhou 1997 Anal Biochem. 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: Life Technologies (former Invitrogen) A36006

Please see the manual provided by the producer: Manual Amplex UltraRed.

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 1:5 with DMSO to get the 2 mM stock solution. Small aliquots (100 µl) of the 2 mM stock solution may be stored at -20°C.

Final concentration

Titrate 20 µl of 2 mM stock solution into the 2 ml O2k-chamber with MiR05 (or MiR05Cr), to obtain a final concentration of 20 µM AmR (you may add 5 µl of the 2 mM stock solution for brief measurements to obtain a final concentration of 5 µM). 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): Prepare a stock solution with 500 U HRP/ml in MiR05 or MiR05Cr; the solution can be used as storage solution at -20 °C. For each measurement, 4 µl of stock solution are titrated into the 2 ml O2k-chamber, the final concentration in the chamber being 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. %; stabilized with acetanilide) = 880 mM H₂O₂.

Prepare fresh, use plastic volumetric flasks:

• Dilution 1 (1:88): 284 µl in 25 ml H2O = 9.9968 mM (= 10 mM for practical purposes).
• Dilution 2 (1:125): 200 µl in 50 ml H2O + 500 µl 1 mM HCl (for stabilization) = 40 µM
• Titrate 2 * 5 µl of the 40 µM stock in 2 ml chamber = 2 * 0.1 µM = total 0.2 µM (= 200 nM) 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, 0.1 µM, 0.2 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).

DatLab (upgraded version) supports automatic H₂O₂ calibration by calculating the calibration Parameters by linear regression and graphical display of the calibration regression:

• Follow point 1.to 7.listed in Features in DatLab 5.2 not yet included in the Manuals.

Since part of the signal change might be caused by an ongoing H₂O₂ production of the biological sample the calibration procedure can be corrected for this "background flux" by including the slope in the calculation.

Therefore

• Select the slope you want to include in the calculation by ticking the appropriate check boxes on the right side of the slope values. If you select more than one slope the mean of all values is used for calculation.
• If for any reaseon the values of the slope displayed do not meet your expectations, e.g. too high value as an artifact due to injection peak,instead of selecting these one you can set a mark on the Amplex slope manually and select this in the window under "Independent slope". This slope will then be used for calculation.

• Finish your calibration with point 8. to 11. of the link mentioned above.

A 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, e.g. cyanide, azide; more information: horseradish peroxidase

• Catalase and other substances consuming or scavanging H₂O₂. The effect of substances in the medium that consume H₂O₂ slowly is taken account of by the calibration procedure. However, such substances decrease the sensitivity of the method. Note that catalase can be a valuable tool for 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 (a strong scavanger of H₂O₂), succinate, (ADP + Mg²⁺), (ATP + Mg²⁺), rotenone, FCCP, oligomycin, antimycin A, malonate, myxothiazol

Superoxide dismutase

Superoxide dismutase (SOD) is optionally included to generate H₂O₂ from superoxide. Results may be compared with and without SOD for evaluation of the contribution of superoxide not endogenously dismutated with the formation of peroxide.

(Sigma-Aldrich S 8409-15KU): Depending on the batch the SOD preparation may contain a specific activity of 2,000–6,000 units/mg protein. We recommend to use the enzyme at 5 U/ml, the final volume to be added to the respiratory chamber has thus to be adjusted accordingly.

Checking for artifacts

The Amplex method is based on the H₂O₂ dependent oxidation of AmR to resorufin by HRP. Under unfavorable conditions AmR may be oxidized even in the absence of H₂O₂. At a small rate such an oxidation occurs in the presence of HRP even without any sample present. The magnitude of this background signal ("drift") depends, among other things, on the light intensity used and can therefore be minimized by using the suggested or lower light intensity. Components of the sample may however induce a far higher, non H₂O₂ related rate of AmR 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 AmR and HRP have to be added to the chamber already containing the sample anyway: First, inject Amplex Red and wait a few minutes for flux stabilization. The Amp slope has 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). Note: The experiment can NOT be continued afterwards!

References