- 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 by cells or mitochondrial preparations in the form of hydrogen peroxide. 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, Hickey 2012 J Comp Physiol B, Fasching 2011 Abstract Berlin
MitoPedia methods:
Fluorometry
Application in HRR
The Amplex Red method[1] can be used with the O2k-Fluorescence LED2-Module[2] using the Fluorescence-Sensor Green. Use black stoppers with black cover-slips to exclude disturbances by external light sources.
The Oxygraph-2k has been coupled to a fluorescence spectrophotometer with a light guide inserted through the black PEEK stopper[3]
Preparation of Amplex® UltraRed solutions
Source: Life Technologies (former Invitrogen) A36006
See the manual provided by the producer: Manual Amplex UltraRed.
Storage solution = stock solution
Prepare a 10 mM storage solution of Amplex® UltraRed reagent (AmR), which will also serve as stock solution, 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. The storage solution should then be divided into 10 µl aliquots and stored 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.
Final AmR concentration
- Titration into into 2 ml O2k-chamber: 2 µl of 10 mM AmR stock solution, final concentration of 10 µM AmR.
You may add 1 µl of the 10 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. Check for any potential effects of the exprimental AmR concentration on respiratory activity of cells or mt-preparations, by titration of AmR after a specific respiratory state has been reached in a control experiment.
Horse radish peroxidase
- Titration into into 2 ml O2k-chamber: 4 µl of HRP stock solution, final concentration 1 U/ml.
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.
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.
Calibration with H2O2
Calibration standards of H2O2: Commercial solution (Sigma-Aldrich 323381 - 25 ml hydrogen peroxide solution 3 wt. %; stabilized with acetanilide, c. 200 ppm) = 880 mM H2O2.
- To prepare a HCl stock solution of 10 µM HCl, take 1 ml of 1 mM HCl storage solution an fill up to 100 ml with distilled water.
- H2O2 dilution 1 (1:88): add 284 µl of a commercial solution of 3 wt.% H2O2 to 20 ml of 10 µM HCl and full up to a final volume of 25 ml with 10 µM HCl, to obtain a 10 mM H2O2 solution.
- H2O2 dilution 2 (1:125): dilute 200 µl of 10 mM H2O2 solution with 10 µM HCl to a volume of 50 ml to obtain the H2O2 stock solution of 40 µM.
- Titration into into 2 ml O2k-chamber: 5 µl of the 40 µM H2O2 stock, step increase of 0.1 µM H2O2. One to three subsequent steps.
Titrations are peformed after addition of respiration medium, HRP and Amplex UltraRed to the O2k chamber. We suggest to perform calibrations at the beginning (sample already present), intermittently at various respiratory states, and near the end of an experiment (but before cytochrome c, catalase or other substances are added that are interfer with the H2O2 measurement).
DatLab supports automatic H2O2 calibration by calculating the calibration parameters by linear regression and graphical display of the calibration regression:
>> | New Features in DatLab 5.2 not yet included in the O2k-Manual.
Since part of the signal change might be caused by an ongoing H2O2 production of the biological sample the calibration can be corrected by including the slope in the calculation.
Therefore
DatLab5 and DatLab 6
- Select the slope you want to include in the calculation (exclude slopes distorted by e.g. the injection of H2O2) by ticking the appropriate check boxes on the right side of the slope values. If you select more than one slope the mark duration weighted mean of all values is used for calculation.
- If for any reason the values of the slope displayed do not meet your expectations, e.g. too high value as an artifact due to the injection peak, instead of selecting you can set a mark on the Amplex slope manually and copy the corresponding value from 'Mark statistics' into the calibration window under "Independent slope". This slope will then be used for calculation.
DatLab 6: In DatLab 6 the slope used for the correction is calculated from a linear regression signal vs time for the selected marks and take from the smoothed slope plot. This eliminates the distortion of the slope by injection artifacts after the Amp signal itself is already stable.
- 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 weighted mean of all values is used for calculation.
all DatLab Versions:
- 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 evaluated small amounts (as typically used in SUIT protocols) of the following substances as 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
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 H2O2 flux (production) is established, a high dose (e.g. 10 µl of a 280,000 U/ml stock solution) of catalase is injected. Catalase competes with HRP for the available H2O2. Then the apparent H2O2 production (the Amp slope) should be reduced to near zero as a control for distinguishing an unspecific chemical background slope from H2O2 dependent Amplex Red oxidation.
Note: The experiment cannot be continued afterwards for measurement of hydrogen peroxide production, whereas respiration can be recorded onwards.
