The cytokine (adiponectin, IL-10, IFN-r, TNF-a, and IL-6) levels in PVAT samples from db/c, db/db exercised, and nonexercised mice were detected with specific antibody using the ELISA kits (BioLegend). The absorbance was measured at 450 nm by using a spectrometer (Bio-tek). Data was expressed as the fold of the level in db/c mice.
A spectrometer
Manufactured by Agilent Technologies
Sourced in United States
A spectrometer is a scientific instrument used to analyze the spectrum of light or other electromagnetic radiation. It separates and measures the different wavelengths of light present in a sample, allowing for the identification and quantification of chemical compounds.
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Lab products found in correlation
Elisa kit, by BioLegend (1 mentions)
Catalase assay kit, by Merck Group (1 mentions)
Thioredoxin reductase assay kit, by Merck Group (1 mentions)
Mouse elisa kit, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Cell counting kit 8 cck 8 reagent, by Vazyme (1 mentions)
Ca 074, by Merck Group (1 mentions)
Z phe arg amc, by Merck Group (1 mentions)
L trans epoxysuccinyl leucylamido 4 guanidino butane e 64, by Merck Group (1 mentions)
Glutathione reductase assay kit, by Merck Group (1 mentions)
Cytochrome c oxidase assay kit, by Merck Group (1 mentions)
15 protocols using a spectrometer
1
Cytokine Profiling in PVAT Samples
2
Catalase Activity Assay Protocol
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Catalase activity was measured using the Catalase Assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. In brief, 25 μl of cytosolic lysate (5~10 μg protein/μl) was mixed with 50 μl of 1x assay buffer and 25 μl of 200 mM H2O2 solution and incubated for 2 min at room temperature. The reaction was stopped by adding a stop solution (15 mM sodium azide in water). Then, 10 μl out of the 100 μl reaction mixture was mixed with 990 μl of the color reagent (150 mM potassium phosphate buffer, pH 7.0, containing 0.25 mM 4-aminoantipyrine and 2 mM 3,5-dichloro-2-hydroxybenzensulfonic acid) in a new tube by inversion. After 15 min of incubation for color development, the absorbance was measured at 520 nm in a spectrometer (Bio-Tek). Activity (μmoles/min/mg protein or U/mg protein) was calculated using the equation “Δμmoles (H2O2) = A520 (Blank)—A520 (Sample).” All samples were run in duplicate.
3
Thioredoxin Reductase Activity Assay
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Thioredoxin reductase activity was measured using the Thioredoxin Reductase Assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. In brief, 10 μl of cytosolic lysate was added to wells in the 96 well plate and then 190 μl of mixture containing 180 μl of working buffer (100 mM potassium phosphate, 10 mM EDTA, and 0.24 mM NADPH), 6 μl of 100 mM DTNB, and 4 μl of either 1 x assay buffer (100 mM potassium phosphate, pH 7.0, 10 mM EDTA) or thioredoxin reductase inhibitor was added to the wells. The Absorbance was read at 412 nm every 10 s for 2 min in a spectrometer (Bio-Tek) to calculate the activity. All samples were run in duplicate.
4
Quantitative Microbial Adherence Assay
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Mucus was diluted 1:5 in sterile distilled water and a volume of 100 μL was added to each well of 96-well ELISA polystyrene plates (Jet Biofil, Guangzhou, China). Plates were dried in a laminar flow cabinet for 48 h and subsequently filled with 200 μL of TSB-2 per well. V. europaeus CECT 8136 and PC1-11 overnight cultures were diluted 1:100 and incubated at 25 °C for 24 h. Bacterial cells were fixed with methanol (99.8%) stained with 0.5% crystal violet solution (v/v), washed three times with distilled water and the bound dye was eluted with acetic acid (33%, v/v) and measured by optical density at 570 nm in a spectrometer (Biotek, Winooski, VT, USA). Results are means calculated from eight replicates.
5
Glutathione Peroxidase Activity Assay
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Glutathione peroxidase activity was measured using the Glutathione Peroxidase Assay kit (Sigma-Aldrich) according to the manufacturer’s instructions. In brief, 80 μl of cytosolic lysate was added to a well in the 96 well plate and then 120 μl of mixture containing 10 μl of NADPH reagent (5 mM NADPH, 42 mM reduced glutathione, and 10 U/ml glutathione reductase), 50 μl of assay buffer (50 mM Tris-HCl, pH 8.0, 0.5 mM EDTA), and 60 μl of 30 mM tert-butyl hydroperoxide was added to the well. The Absorbance was read at 340 nm every 10 s for 1 min in a spectrometer (Bio-Tek) to calculate the activity. All samples were run in duplicate.
6
Serum Cytokine Quantification in LPS-Induced Mice
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Briefly, blood was collected from the heart 3 h after LPS administration (n = 5 per group). Serum was collected by centrifugation at 1700g 4°C for 15 min. Serum IL-6 and TNF-α levels were analyzed using mouse ELISA kits according to the manufacturer's protocol (Thermo Fisher Scientific) at 450 nm with a spectrometer (BioTek).
7
DPPH Radical Scavenging Assay Protocol
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The method developed by Sharififar et al. (2009) was followed. Briefly, 180 μl of the 2,2-diphenyl-1-picrylhydrazyl (DPPH; 167 μM in ethanol) reaction solution was mixed with 20 μl of samples, and then incubated in a dark environment for 30 min, and the absorbance was measured at 520 nm using a spectrometer (BioTek). As a control, DPPH solution in ethanol was mixed with same volume of ethanol, and the absorbance of the solution was measured. IC50 values of the DPPH radical scavenging activity were calculated, and all experiments were conducted in triplicate.
8
ABTS Radical Scavenging Assay Protocol
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The method modified by Muanda et al. (2011) was followed. Briefly, a 7 mM 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was mixed with 2.45 mM diammonium salt in phosphate buffered saline (PBS) solution (100 mM, pH 7.4), and then left in a dark environment for 16 h. The reaction solution was diluted with ethanol so that the absorbance value is 0.7 ± 0.05 at 734 nm before use. 20 μl of samples was treated with 180 μl of ABTS solution, incubated for 10 min in a dark environment, and the absorbance was measured at 734 nm using a spectrometer (BioTek). 50% inhibitory concentration (IC50) values of the ABTS radical scavenging activity were calculated, and all experiments were conducted in triplicate.
9
MTT Assay for Cell Viability
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Cell viability was examined by using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT; Sigma-Aldrich) assay. RAW264.7 cells were seeded in a 24-well plate at a density of 2 × 105 cells/well and incubated for 24 h. Furthermore, cells were treated with BEN815 (6.25, 12.5, 25, 50, and 100 μg/ml) along with 100 ng/ml LPS (Sigma-Aldrich) for 20 h. After that, the MTT reagent (0.5 mg/ml) was added, and after 4 h the formazan crystals formed in the cells were dissolved with dimethyl sulfoxide (DMSO); the absorbance was measured at 570 nm using a spectrometer (BioTek, Winooski, VT, USA).
10
WST-1 Cell Viability Assay
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Cells were seeded in RPMI + 10% FBS at 5 × 103 cells/well in 96-well plate for 24 h. Cells were then washed with RPMI + 0.1% BSA and synchronized by serum deprivation in RPMI + 0.1% BSA for 24 h before treatment with an indicated reagent for 48 h. Following the treatment, the WST-1 reagent was added to each well. Cells were incubated for 4 h before measurement at 450 nm and 690 nm (reference) with a spectrometer (Biotek). After subtracting blank control, absorbance at 690 nm was subtracted from that obtained at 450 nm. The viability of the cells was calculated as a percentage of cell viability compared to control (untreated cells) using the following equation: % Viability = 100 × (A450–A690)treated/(A450–A690)control; A, absorbance.
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