Mice heterozygous for a disrupted Uox transgene (Wu et al.,1994 (link)) were obtained from Jackson Laboratories (Bar Harbor, ME) and were bred to generate the mice used in this study. The genetic background of the mice is C57BL/6J and 129Sv. Mice were maintained according to National Institutes of Health guidelines on a standard diet with free access to water. To limit the levels of hyperuricemia, the UOX−/− mice in the longevity study received water supplemented with allopurinol (0.67 mM from birth to weaning, 0.18 mM for the first year, and 0.26 mM thereafter). Separate groups of mice were used for behavior, exercise, and stroke studies. Hyperuricemic conditions were obtained by terminating the allopurinol therapy 2 weeks before the beginning of the experiment. All procedures on animals were approved by the Animal Care and Use Committee of the National Institute on Aging Intramural Research Program. Blood was collected either by retro-orbital bleeding in live mice or by cardiac puncture at the time of euthanasia. Serum UA concentrations were measured using an assay kit (Randox, San Diego, CA) on a Roche Cobas Fara II robotic chemical analyzer according to the manufacturer’s specifications. DC-Cal and DC-Trol Level 1 and 2 Controls (Diagnostic Chemicals Limited, Oxford, CT) were used for instrument calibration and interassay accuracy, respectively.
Cobas fara 2
Manufactured by Roche
Sourced in Switzerland
The Cobas Fara II is a clinical chemistry analyzer developed by Roche. It is designed to perform automated, high-throughput analysis of various biochemical parameters in clinical samples. The Cobas Fara II can handle a variety of sample types and provides accurate and reliable results to support clinical decision-making.
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12 protocols using cobas fara 2
1
Hyperuricemic Mouse Model Protocol
2
Hematology and Iron Metabolism Assessment
3
Assessing Cell Viability and Function
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In order to assess the cell functions and viability, 200 μL of media were withdrawn from the mixing chamber at fixed intervals (0, 4, and 24 h). Biochemical assays were performed to measure the change over time in the metabolite and pro-inflammatory marker levels in the media.
Free Fatty Acids (FFAs) and triglycerides were measured by a colorimetric enzyme assays (respectively NEFA C test-Wako Chemicals GmbH, Germany and Hagen Diagnostica SRL, S. Giovanni V.no (AR), ITALY). Glycerol was determined by a modified Lloyd assay using an automated spectrophotometer Cobas Fara II (Roche) [27 (link)]. IL-6, (eBioscience Dx Diagnostic, Vienna, Austria), E-Selectin, (Boster Biological Technology, LDT, Tema Ricerca, Bologna, Italy), MCP-1 (Life Technologies Italia, Monza-MI, Italy, albumin (Bethyl Laboratories, Montgomery, TX, USA), were determined using ELISA. Other assays and their results are reported in the Supporting Information,S1 File .
At the end of the experiments the cells were fixed and stained with DAPI and phallodin or anti-vWF (all from Thermo Fischer, Italy) and observed with a confocal (Nikon A1, IT) or fluorescent microscope (Olympus X81, IT). Adipose tissue was stained with oil-red stain. The intensity of vWF staining was quantified by image processing as described in the Supporting Information,S1 File .
Free Fatty Acids (FFAs) and triglycerides were measured by a colorimetric enzyme assays (respectively NEFA C test-Wako Chemicals GmbH, Germany and Hagen Diagnostica SRL, S. Giovanni V.no (AR), ITALY). Glycerol was determined by a modified Lloyd assay using an automated spectrophotometer Cobas Fara II (Roche) [27 (link)]. IL-6, (eBioscience Dx Diagnostic, Vienna, Austria), E-Selectin, (Boster Biological Technology, LDT, Tema Ricerca, Bologna, Italy), MCP-1 (Life Technologies Italia, Monza-MI, Italy, albumin (Bethyl Laboratories, Montgomery, TX, USA), were determined using ELISA. Other assays and their results are reported in the Supporting Information,
At the end of the experiments the cells were fixed and stained with DAPI and phallodin or anti-vWF (all from Thermo Fischer, Italy) and observed with a confocal (Nikon A1, IT) or fluorescent microscope (Olympus X81, IT). Adipose tissue was stained with oil-red stain. The intensity of vWF staining was quantified by image processing as described in the Supporting Information,
4
Oxidative Stress Biomarkers in Tissues
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Homogenates of liver and ovary tissues, prepared as described by Zhang et al. [5 (link)], were clarified by centrifugation at 3000× g for 10 min at 4 °C and the supernatants were used in enzyme assays. In serum, liver and ovary samples, content of malondialdehyde (MDA), total antioxidant capacity (T-AOC), and glutathione (GSH) were determined. The activity of glutathione peroxidase (GSH-PX), copper-Zn superoxide dismutase (CuZnSOD), and alkaline phosphatase (AKP) were measured using kits purchased from Nanjing Jiancheng Bioengineering Institute, Nanjing, China, and using an automated spectrophotometric analyzer (Cobas FARA II, Roche, Palo Alto, CA, USA) according to the manufacturer’s directions.
5
Plasma Metabolite Analysis Protocol
6
Plasma Lipid Profile Analysis
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Plasma concentrations of total cholesterol, HDL-C, triglycerides, and apolipoproteins were measured using blood samples obtained after a minimum of an 8-h fast using CDC-standardized methods. Plasma total cholesterol (TC), HDL-C, and triglycerides were measured enzymatically on a Cobas Fara II autoanalyzer (Roche Diagnostic Systems Inc., Basel, Switzerland) using Sigma reagents (Sigma Chemical Co. St Louis, MO, USA). Apolipoproteins were measured turbidimetrically using Wako reagents (Wako Chemicals USA Inc., Richmond, VA, USA). Measurements were performed on frozen (−80 °C) EDTA plasma and serum. LDL-C was calculated using the Friedewald equation.
7
Quantifying MPO Activity in Tissues
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Intestinal and lung tissue segments were homogenized and processed in 800 µl of an equivalent volume of 50 mmol/L phosphate-buffered saline (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide. Tissue segments were sonicated at 40 W for 1 minute. Samples were centrifuged at 21,000xg for 20 min at 4°C and supernatants were collected. The protein content of all the samples was determined using the Pierce BCA Protein Assay (Pierce, Rockford, IL). MPO activity was assessed by adding 25 µL of 3 mM H2O2 as previously described [16] (link). Change in absorbance was measured using a Cobas FARA II centrifuge analyzer (Roche Diagnostic Systems, Montclair, NJ) at A655 nm over 3 minutes duration. MPO activity was quantified and data displayed as Units/mg of total protein.
8
Serum Biomarkers of Hepatic Injury
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Analysis was performed on baseline serum samples and the single serum samples containing the highest value for ALT level (i.e., the only post-baseline serum sample archived and therefore available for analysis). Serum concentrations of glutamate dehydrogenase (GLDH; Randox Laboratories, Kearneysville, WV) and sorbitol dehydrogenase (SDH; Genzyme Diagnostics, Cambridge, MA) were determined using a commercial kit by quantifying the decrease in absorbance according to the manufacturer’s protocol. Assay results for both GLDH and SDH were quantified using the Cobas Fara II clinical chemistry analyzer (Roche Diagnostics, Indianapolis, IN). Caspase-cleaved and total K18 were determined using the M65 and M30 (apoptosense) ELISAs, respectively, in accordance with the manufacturer’s guidelines (Peviva, West Chester, OH). Total HMGB1 content was determined in serum by enzyme-linked immunosorbent assay (ELISA) per the manufacturer’s protocol (IBL International, Hamburg, Germany). For all ELISA analytes, the inter- and intra-assay variability was <10%.
9
Metabolic Responses to Fasting and Exercise
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Sedentary mice were either fed ad libitum, and blood was collected at ZT14 (“fed ZT14”), or fasted for 4 hr late in the afternoon (ZT7–ZT11), and blood was collected at ZT11 (“fasted ZT11”). A separate cohort of endurance-trained WT and mKO littermates was measured after 4 wk training on a motorized treadmill 25 cm/s and 0% incline 1 hr daily, starting around ZT12. Blood was collected after running 25 cm/s and 0% incline for 1 hr. Blood was collected from the orbital sinus in heparin-coated Pasteur pipettes and centrifuged immediately after collection. Plasma samples were kept at −20 °C until dosing. FFAs, β-OH-B, and AcAc were dosed using an automated spectrophotometer, Cobas Fara II (Roche), according to the manufacturer’s instructions. Blood glucose and lactate levels were measured with a YSI 2300 STAT Plus glucose and lactate analyzer (YSI Life Sciences, Yellow Springs, OH) according to the manufacturer’s instructions.
10
Cyanide-insensitive Palmitoyl-CoA Oxidase Assay
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Cyanide-insensitive palmitoyl-CoA oxidase was determined essentially according to the method by Lazarow (1981 (link)), specifically as follows:
Five µL of the liver cytosol (prepared as described in the preceding chapter “Aryl hydrocarbon receptor”) derived from the NVP-treated (and untreated control) male rats were mixed in a centrifugal analyzer Cobas Fara II (Hoffmann La Roche) with 250 µL reaction mixture consisting of 28.2 mL 50 mM Tris buffer pH 8.01, 150 µL 2% Triton-X-100, 300 µL 1 mM FAD, 300 µL 10 mM CoA, 150 µL 1.5% BSA, 90 µL 0.33 mM dithiothreitol, 300 µL 20 mM NAD and after homogenization 300 µL 100 mM KCN. The reaction was started by adding 5 µL of 5.03 µM palmitoyl-CoA aqueous solution. The increase of the absorption at ʎ = 334 nm due to NADH formation was determined and translated to palmitoyl-CoA oxidase U/L by the Cobas Fara II software.
Five µL of the liver cytosol (prepared as described in the preceding chapter “Aryl hydrocarbon receptor”) derived from the NVP-treated (and untreated control) male rats were mixed in a centrifugal analyzer Cobas Fara II (Hoffmann La Roche) with 250 µL reaction mixture consisting of 28.2 mL 50 mM Tris buffer pH 8.01, 150 µL 2% Triton-X-100, 300 µL 1 mM FAD, 300 µL 10 mM CoA, 150 µL 1.5% BSA, 90 µL 0.33 mM dithiothreitol, 300 µL 20 mM NAD and after homogenization 300 µL 100 mM KCN. The reaction was started by adding 5 µL of 5.03 µM palmitoyl-CoA aqueous solution. The increase of the absorption at ʎ = 334 nm due to NADH formation was determined and translated to palmitoyl-CoA oxidase U/L by the Cobas Fara II software.
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