For expression assays, 3T3‐L1 cells, human adipocytes, and HEK-293 AD cells, were transfected with the corresponding plasmid vectors at 2.5 µg/mL using a 7.5:1000 dilution of Lipofectamine 2000 (Invitrogen), and cultured for 48 h prior to the experiments. For silencing studies, cells were transfected with a 7.5:1000 dilution of Lipofetamine RNAiMAX (Invitrogen) and mouse Rab34 siRNA (Dharmacon), mouse UBA1 siRNA (Dharmacon), or control siRNA (Sigma-Aldrich) (scrambled-transfected cells) at 25 nmol/L. Then, cells were kept in culture for 72 h. At the end of the experiments, cells were processed for confocal microscopy and/or immunoblotting as indicated in the corresponding sections. In another set of experiments, cells were collected in radioimmunoprecipitation assay (RIPA) buffer and intracellular concentration of TGs was determined using Triglyceride Reagent (Sigma-Aldrich) and Amplex UltraRed Reagent (Invitrogen), while culture media were analyzed for free glycerol content using Amplex UltraRed Reagent (Invitrogen) and Free Glycerol Reagent (Sigma-Aldrich) as previously described [24 (link)].
Amplex ultrared reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
Amplex UltraRed reagent is a fluorogenic substrate for the detection and quantification of hydrogen peroxide (H2O2) and other oxidants. It is used in analytical and research applications to measure oxidative processes.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase, by Merck Group (5 mentions)
Fluorescence led2 module, by Oroboros (3 mentions)
Bca kit, by Thermo Fisher Scientific (3 mentions)
Dc300 ccd camera, by Leica (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Horseradish peroxidase, by Roche (2 mentions)
Rf 5301, by Shimadzu (2 mentions)
Dfc420 camera, by Leica (1 mentions)
Trehalose dihydrate, by Merck Group (1 mentions)
Igg alex fluor 647, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline ph 7.4, by Thermo Fisher Scientific (1 mentions)
Lipase b acrylic resin, by Merck Group (1 mentions)
α chymotrypsin from bovine pancreas, by Merck Group (1 mentions)
D glucose anhydrous, by Merck Group (1 mentions)
N succinyl ala ala pro phe p nitroanilide, by Merck Group (1 mentions)
3 3 5 5 tetramethylbenzidine tmb liquid substrate system for elisa, by Merck Group (1 mentions)
Fluostar omega, by BMG Labtech (1 mentions)
Pyruvate, by Merck Group (1 mentions)
Hemin, by Merck Group (1 mentions)
Methyl viologen, by Merck Group (1 mentions)
52 protocols using amplex ultrared reagent
1
Lipofectamine-mediated Transfection and Silencing in Adipocytes
2
Evaluating Antipsychotic Impact on hDDO
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The effect of antipsychotic drugs on hDDO activity was evaluated in vitro using a coupled enzyme assay and the Amplex UltraRed reagent (Life Technologies, Carlsbad, CA USA). Detailed procedure is described in Supplementary Methods .
3
Microscopic Visualization of Root Oxidative Stress
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Ten roots of comparable size and length, belonging to plantlets randomly selected for each treatment, were isolated and sectioned with hand microtome, 3-4 mm from the root tip, to explore possible distribution of nano PS aggregates in root tissues with light microscope (Leitz Diaplan Wetzlar, Germany, equipped with a Leica DFC 420 camera, Leica Microsystems, Germany). Amplex UltraRed Reagent (Life Technologies, USA) was applied to cross sections for in situ detection of H 2 O 2 (Ruffini Castiglione et al., 2016) . After staining, slices were mounted in glycerol and observed with fluorescence microscope (568ex/681em nm). BODIPY 581/591 C11 was applied as a fluorescent marker to visualize lipid peroxidation levels with a change of the fluorescence emission peak from red to green (Ruffini Castiglione et al., 2016) . Microscope analysis was performed acquiring simultaneously the green (485ex/510em nm) and the red fluorescence (581ex/591em nm) signals and merging the two images (Kovácik et al., 2014) . Fluorescence microscope analysis was carried out with a Leica DMLB, equipped with appropriate set of excitation/emission filters and with a Leica DC300 ccd camera.
4
Enzymatic Activity Assays Development
5
Nematode Oxidative Stress Assay
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Live nematodes were dispensed into wells of a 96‐well plate containing Amplex UltraRed reagent (Life Technologies, final concentration 0.1 μM in M9 buffer). Samples were incubated at 25°C for 3 h, thus fluorescence was read in a BMG LABTECH FLUOstar Omega plate reader at excitation 544 nm and emission 590 nm. Data were normalized based on the total proteins content of each sample. The data presented as mean ± SEM were tested for significance by the nonparametric Mann–Whitney test, using GraphPad Prism. Significant results were marked according to critical p‐values: ***p < 0.001; **p < 0.01; *p < 0.05.
6
Multidisciplinary Biochemical Protocols
7
Amplex Red Assay for Superoxide and Hydrogen Peroxide
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Amplex™ UltraRed Reagent (Life technologies) was used to measure superoxide plus hydrogen peroxide in cells. Briefly, NRVMs were plated on black 96-well microplates. Forty-eight hours post lactate treatment, cells were rinsed one time with phosphate-buffered saline (PBS). A solution containing Amplex Red dye (final concentration: 50 microM), hydrogen peroxide (0.0015% final) and superoxide dismutase (SOD, final concentration: 5 units/mL) was added to each well to a final volume of 200 microL and incubated in the dark at 37°C for 30 mins. Sample fluorescence was measured using a fluorescence platereader (iD5, Molecular Devices) at excitation and emission wavelengths of 540 and 600 nm, respectively. The cells were washed twice with 100 microL PBS and cell number was assessed using CyQuant cell proliferation assay kit according to the manufacturer's instructions (ThermoFisher). Sample fluorescence was measured using the same plate reader at excitation and emission wavelengths of 580 and 527 nm, respectively. Each condition was examined in 15 wells per plate in 4 different NRVM preparations leaving the edges free of cells. Data shown is from a single representative experiment.
8
Root Oxidative Stress Assessment
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Five roots of comparable size and length, randomly selected from control and treated plants, were excised and sectioned with hand microtome in correspondence to the initial root hair area. Cross sections were immediately processed with fluorescent probes specific for hydrogen peroxide and lipid peroxidation. Fluorescent Amplex UltraRed Reagent (Life Technologies, USA) was applied for in situ detection of H 2 O 2 following manufacturing instructions and protocol reported in Ruffini Castiglione et al. (2016) . After staining, slices were mounted in glycerol and observed with fluorescence microscope (568ex/681em nm).
BODIPY 581/591 C11 was used as free radical sensor to visualize lipid peroxidation levels as a change of the fluorescence emission peak from red to green. The slices were incubated and stained following a previous protocol (Ruffini Castiglione et al. 2016) . Microscope evaluation was performed acquiring simultaneously the green (485ex/510em nm) and the red fluorescence (581ex/ 591em nm) signals and merging the two images (Kovácik et al. 2014) .
Fluorescence microscope analysis was carried out with a Leica DMLB, equipped with appropriate set of excitation/emission filters and with a Leica DC300 ccd camera.
BODIPY 581/591 C11 was used as free radical sensor to visualize lipid peroxidation levels as a change of the fluorescence emission peak from red to green. The slices were incubated and stained following a previous protocol (Ruffini Castiglione et al. 2016) . Microscope evaluation was performed acquiring simultaneously the green (485ex/510em nm) and the red fluorescence (581ex/ 591em nm) signals and merging the two images (Kovácik et al. 2014) .
Fluorescence microscope analysis was carried out with a Leica DMLB, equipped with appropriate set of excitation/emission filters and with a Leica DC300 ccd camera.
9
In Situ Localization of Oxidative Stress
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Six roots of comparable growth level, randomly selected from control and treated plants, were excised and sectioned with hand microtome right above the differentiation zone. At least 10 cross sections for each root were immediately processed with fluorescent probes specific for in situ localization of hydrogen peroxide and oxidative damage in cells and membranes. Amplex UltraRed Reagent (Life Technologies, USA) was applied for in situ detection of H 2 O 2 following the protocol reported in Ruffini Castiglione et al. ( 2016). After staining, slices were mounted in glycerol and observed with fluorescence microscope (568ex/681em nm). BODIPY 581/591 C11 was used as free radical sensor to visualize lipid peroxidation levels as a change of the fluorescence emission peak from red to green. The slices were incubated and stained following a previous protocol (Ruffini Castiglione et al., 2016) . Microscope evaluation was performed acquiring simultaneously the green (485ex/510em nm) and the red fluorescence (581ex/591em nm) signals and merging the two images (Kováčik et al., 2014) . Fluorescence microscope analysis was carried out with a Leica DMLB, equipped with appropriate set of excitation/emission filters and with a Leica DC300 ccd camera.
10
Hydroxyapatite Disc Assay Optimization
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