Liquid analysis of oxRilac solutions was performed after one freeze–thaw cycle. Changes in pH were determined using a pH meter (Mettler Toledo, Giessen, Germany). Hydrogen peroxide (H2O2) was quantified using the Amplex UltraRed Assay (ThermoFisher Scientific, Bremen, Germany) according to the supplier’s instructions. Fluorescence was measured at λex 530 nm and λem 590 nm using a microplate reader (F200; Tecan, Männedorf, Switzerland). Concentrations were calculated against a standard curve.
Amplex ultrared assay
Manufactured by Thermo Fisher Scientific
Sourced in United States
Amplex UltraRed Assay is a fluorometric detection kit for detecting and quantifying hydrogen peroxide (H2O2) and peroxidase activity. The kit uses the Amplex UltraRed reagent, which reacts with H2O2 in the presence of horseradish peroxidase (HRP) to produce a highly fluorescent product. The resulting fluorescence can be measured using a fluorescence microplate reader.
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Lab products found in correlation
Ph meter, by Mettler Toledo (1 mentions)
Griess assay, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase, by Merck Group (1 mentions)
Speedvac, by Thermo Fisher Scientific (1 mentions)
Envision xcite multimode plate reader, by PerkinElmer (1 mentions)
Spectramax m2, by Molecular Devices (1 mentions)
5 protocols using amplex ultrared assay
1
Analyzing Oxidative Stress in oxRilac
2
Quantifying Reactive Species in Plasma
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To quantify hydrogen peroxide (H2O2), one of the final products of the short-lived reactive species chemistry generated by the gas plasma, the amplex ultra red assay (ThermoFisher, Waltham, Massachusetts, USA), was used according to the manufacturer’s instructions. For the detection of two other stable molecules, nitrite (NO2-) and nitrate (NO3-), the Griess assay (ThermoFisher, Waltham, Massachusetts, USA) was performed according to the manufacturer’s instructions. As liquid carriers, PBS and cell culture media were used. Quantification was performed by comparing to standards with known H2O2, NO2- and NO3- concentrations. The ROS/RNS quantification assays were performed under equal CCP treatment regimes as for treatment of cells, i.e., 200 µL in 48-well plates.
3
Quantification of LOXL2 Enzymatic Activity
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LOXL2 activity was measured using the Amplex UltraRed assay (Thermo Fisher Scientific) as previously described in Trackman and Bais (3 (link)). For this, 4 µg LOXL2 was assayed in 50 mM Na borate, pH 8.2 in the presence of 10 mM cadaverin (MilliporeSigma), 10 µM Amplex UltraRed, and 1 U/ml horseradish peroxidase (MilliporeSigma). LOXL2 activity was also measured using 1.25 µM laminin or 1.65 µM TE. Resorufin fluorescence was measured at 37°C for 60 min using an EnVision Xcite multimode plate reader (PerkinElmer, Waltham, MA, USA) at λex = 535 nm and λem = 610 nm. The amount of H2O2 released by LOXL2 was determined using a standard curve of hydrogen peroxide. β-Aminopropionitrile (β-APN) (500 µM) was added to the reaction mixture in some experiments. Measurements were performed in triplicate, and a representative experiment is shown.
TE was dissolved in 50 mM Na borate buffer, pH 8.0 at a concentration of 10 mg/ml, then incubated at 50°C for 15 min to induce coacervation. LOXL2 was then added at an enzyme:substrate ratio of 1:500 (w/w), and the mixture of enzyme and substrate was incubated at 50°C for 24 h. The precipitated insoluble cTE that had formed was washed with a solution of water and ethanol (1:1, v/v), dried in a SpeedVac (Thermo Fisher Scientific), and stored at −26°C prior to further analysis.
TE was dissolved in 50 mM Na borate buffer, pH 8.0 at a concentration of 10 mg/ml, then incubated at 50°C for 15 min to induce coacervation. LOXL2 was then added at an enzyme:substrate ratio of 1:500 (w/w), and the mixture of enzyme and substrate was incubated at 50°C for 24 h. The precipitated insoluble cTE that had formed was washed with a solution of water and ethanol (1:1, v/v), dried in a SpeedVac (Thermo Fisher Scientific), and stored at −26°C prior to further analysis.
4
Argon Plasma Treatment of 3D Melanoma Spheroids
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The plasma jet kINPen MED (neoplas tools, Greifswald, Germany) was used to generate plasma at room temperature and ambient conditions. Argon (purity 99.999%; Air Liquide, Düsseldorf, Germany) was used as the carrier gas, which was ignited by applying a high voltage of 2–3 kV and a frequency of 1 MHz. During plasma generation, a pulsed mode with a frequency of 2.5 kHz was applied. The gas flow was set to three standard liters per minute. In order to modify the ambient conditions, a shielding device made of glass was employed. This way, the plasma effluent was well separated from the surrounding room air. The shielding gas consisted of different ratios of nitrogen and oxygen, starting from 0% O2 and 100% N2 going up in 5 steps to 100% O2 and 0% N2. In this study, the plasma was applied either directly to the cultured spheroids in the 96-well plate (direct treatment) or to cell culture medium that was then added to the wells of the 96-well plate containing 3D melanoma spheroids (indirect treatment mode). After plasma treatment, the medium was not changed. The particular treatment modality is shown schematically on the top of each figure. To quantify the amount of hydrogen peroxide (H2O2) that was introduced into the liquids, the Amplex Ultra Red Assay (Thermo Scientific, Waltham, MA, USA) was utilized as previously described [34 (link)].
5
Mitochondrial ROS Production Assay
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Mitochondrial O2•−/H2O2 release was assessed using the Amplex Ultra Red assay (AUR; Thermo Fisher Scientific Inc.) according to a previous report [28 (link)]. Cardiac mitochondria were diluted to 0.1 mg/mL in MESH buffer and added to the wells of a black 96-well plate. AUR reagent was then added to each well at a final concentration of 10 μM. Reactions were started by adding 50 μM 2-oxoglutarate and 50 μM pyruvate, and 50 μM malate was added to complete the Krebs cycle, ensuring the full oxidation of 2-oxoglutarate to measure the ROS releases by OGDHc. Changes in fluorescence were monitored at the excitation and emission wavelengths of 565 nm and 600 nm, respectively, using a Spectramax M2 microplate reader (Molecular Devices, Shanghai, China) for 5 min.
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