![]() ![]() Horseradish peroxidase Titration into into 2 mL O2k-chamber: 4 µL of HRP stock solution, final concentration 1 U/mL. As this chemical has a pink color, it is normal for the medium to have a pink color at the end of the experiment. During the experimental run, the product UltroxRed (or resorufin), generated from the Amplex UltraRed (or Amplex Red) plus H 2O 2 in the reaction catalyzed by horseradish peroxidase, accumulates in the chamber. The appearance of pink coloring may indicate that the chemical has been compromised. Color of AmR and the medium Amplex UltraRed freshly dissolved should not present any color. Check for any potential effects of the experimental 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. The final concentration of AmR becomes diminished during an experiment due to AmR consumption and dilution by titrations in a SUIT protocol. These determine the total AmR consumption by H 2O 2 production during the experiment (check by initial and final H 2O 2 titrations for calibration). Optimize the final AmR concentration according to respiration media, sample type and concentration, and experimental protocol. You may add 1 µL of the 10 mM stock solution for brief measurements to obtain a final concentration of 5 µM. AmR, therefore, should be used at the lowest concentration compatible with the total H 2O 2 production in an experimental run, including calibrations and chemical background conversion of AmR (Makrecka-Kuka et al 2015 Komlodi et al 2018). AmR, similar to other chemical probes, may exert an inhibitory effect on mitochondrial and cell respiration. » O2k manual titrations MiPNet09.12 O2k-Titrations Titration into 2 mL O2k-chamber: 2 µL of 10 mM AmR stock solution, final concentration of 10 µM AmR. 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# 11), known as Amplex® Red. Note: The preparation of the 10 mM storage solution follows the procedure suggested by the manufacturer. We have observed that after long-term storage AmR may exert a profound inhibitory effect on cell respiration. When stored properly, the 10 mM storage solution is stable for 6 months. The storage solution should then be divided into 20 µL aliquots and stored in the dark in a desiccator, protected from moisture at –20☌ for future use. Storage solution = stock solution Prepare a 10 mM storage solution of Amplex® UltraRed reagent (AmR), which will also serve as a stock solution, by adding 340 μL of fresh, high‑quality DMSO (Sigma D8418) to one commercial vial of Amplex® UltraRed reagent (1 mg). Preparation of 10 mM stock solution (dissolved in DMSO) - see also Manual Amplex® UltraRed by Life Technologies. AmR reacts with H 2O 2 catalysed by horseradish peroxidase.Īpplication in HRR Amplex® UltraRed AmR: Amplex® UltraRed from Life Technologies (former Invitrogen): A36006 6* 1 mg commercial vials, store at -20☌.The use of AmR in HRR to simultaneously determine respiration and H 2O 2 flux has been intensively tested in the Oroboros lab (see references). The O2k has also been coupled to a fluorescence spectrophotometer with a light guide inserted through the black PEEK stopper (Anderson 2011). The fluorescence sensors are inserted though the windows of the O2k-Chambers (Hickey et al 2012). Use black stoppers with black cover-slips to exclude disturbances by external light sources. ![]() 13.6 How to connect the Smart Fluo-Sensors to the O2k?Īmplex Red or Amplex UltraRed in research articles CitationĪmplex® UltraRed in O2k-FluoRespirometry O2k-Fluo Smart-Module and O2k-Fluo LED2-Module In high-resolution respirometry HRR the Amplex® UltraRed method (Mohanty et al 1997) is used with the O2k-FluoRespirometer selecting the Fluorescence-Sensor Green (O2k-Series D-G: O2k-Fluo LED2-Module) and Smart Fluo-Sensor for O2k-Series H - I.13.5 Exchange of filters in the fluorescence sensors.13.3 Data analysis with the Excel template.13.1 Set marks to calibrate the fluorescence signal using Amplex® UltraRed assay.10 H 2O 2 flux analysis and mark setting.6.4 Substances incompatible with the Amplex® UltraRed assay.4.3 HRP-independent background AmR flux.4.2 Calculation of background fluorescence slope of MiR05.4.1 Experimental media for the AmR assay.2.1 O2k-Fluo Smart-Module and O2k-Fluo LED2-Module.2 Amplex® UltraRed in O2k-FluoRespirometry.1.1 Amplex Red or Amplex UltraRed in research articles. ![]()
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