Glucose tolerance test (GTT), pyruvate tolerance test (PTT), insulin tolerance test (ITT) and Intralipid tolerance test were performed as previously described43 (link)–46 . Serum insulin levels were measured using ALPCO Mouse Insulin ELISA Jumbo kit (Cat. Number: 80-INSMS-E10). β3 adrenergic receptor agonist CL 316,243 (Sigma, Ca. Number: C5976) was used at 1 mg/kg body weight through intraperitoneal injection, CL 316,243-stimulated glycerol release was performed as previously described44 (link). Liver triglyceride extraction and quantification was performed at UTSW metabolic phenotyping core using a previously published protocol43 (link). Serum parameters (aspartate transaminase, alanine transaminase, cholesterol, triglyceride, very low-density lipoprotein, low-density protein and direct high density lipoprotein) were measured and calculated with a VITROS analyzer (Ortho Clinical Diagnostics) at UTSW metabolic phenotyping core.
Vitros analyzer
Manufactured by Ortho Clinical Diagnostics
The VITROS analyzer is a diagnostic instrument designed for automated clinical chemistry and immunoassay testing. It is used in laboratory settings to perform a variety of diagnostic tests on patient samples.
Automatically generated - may contain errors
Lab products found in correlation
Cl316 243, by Merck Group (1 mentions)
Glycogen colorimetric fluorometric assay kit, by Abcam (1 mentions)
Mouse rat igf 1 quantikine elisa kit, by R&D Systems (1 mentions)
Edta coated vacutainer tubes, by BD (1 mentions)
Aprotinin, by Merck Group (1 mentions)
Ezrmgh 45k, by Merck Group (1 mentions)
Ezrgra 90k, by Merck Group (1 mentions)
P hydroxymercuribenzoic acid, by Merck Group (1 mentions)
Biotek power wave xs microplate spectrophotometer, by Agilent Technologies (1 mentions)
Tunel kit, by Merck Group (1 mentions)
Anti f4 80, by Merck Group (1 mentions)
Anti α sma, by Abcam (1 mentions)
Anti myeloperoxidase, by Abcam (1 mentions)
8 protocols using vitros analyzer
1
Metabolic Tolerance Tests and Analyses
2
Metabolic Evaluation of Liver Function
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Glucose tolerance test (GTT), pyruvate tolerance test, insulin tolerance test (ITT), and triglyceride clearance test were performed as previously described (17 (link)–19 (link)). Liver triglyceride extraction and quantification were performed at the UTSW Metabolic Phenotyping Core using a previously published protocol (17 (link)). Liver glycogen was extracted in water and assayed with a protocol provided in the Glycogen Colorimetric/Fluorometric Assay Kit from BioVision (catalog no. K646-100). Serum parameters (albumin, bilirubin, aspartate transaminase [AST], alanine transaminase [ALT], cholesterol, triglyceride, VLDL, LDL, and direct HDL) were measured and calculated with a VITROS analyzer (Ortho Clinical Diagnostics).
3
Metabolic Phenotyping of Mice
4
Biomarker Quantification in Fed Mice
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Blood was taken from fed animals in the morning and was centrifuged at 8000 g for 5 min, and then the supernatants were collected for multiple analyses. Adiponectin was measured using an ELISA kit from Invitrogen (Cat# EZMADP-60K). Serum insulin levels were measured using ALPCO Mouse Insulin ELISA Jumbo kit (Cat# 80-INSMS-E10, Mercodia Developing Diagnostic). Serum IGF-1 levels were measured by Mouse/Rat IGF-1 Quantikine ELISA kit (R and D Systems, Inc, Minneapolis, MN). Corticosterone was measured using a Corticosterone Competitive ELISA Kit (Cat# EIACORT); serum parameters were measured and calculated with a VITROS analyzer (Ortho Clinical Diagnostics) at UTSW metabolic core.
5
Serum Biomarker Analysis Protocol
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Serum parameters (aspartate transaminase, alanine transaminase, cholesterol, triglyceride, very low-density lipoprotein, low-density protein, and direct high-density lipoprotein) were measured and calculated with a VITROS analyzer (Ortho Clinical Diagnostics) at the Metabolic Phenotyping Core at UT Southwestern. Mouse serum adiponectin levels were measured using ELISA kits from Millipore (Cat# EZMADP-60K), mouse serum leptin levels were measured using ELISA kits from Sigma–Aldrich (Cat. # GF050).
6
Serum Uric Acid and Knee Pain
7
Plasma Biomarker Extraction and Quantification
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Blood was collected into 3 mL ice-cold EDTA-coated vacutainer tubes (BD Biosciences, Franklin Lakes, NJ) and aliquoted into microfuge tubes containing either the protease inhibitors p-hydroxymercuribenzoic acid (Sigma Aldrich; final concentration 1 mM; used for ghrelin measurement) or aprotinin (Sigma Aldrich; final concentration 250 KIU/mL; used for LEAP2 measurement) or lacking protease inhibitor (used for GH, IGF-1, liver enzymes, total cholesterol, and triglycerides). The samples were immediately centrifuged at 4 °C at 1,500 g for 15 min. To stabilize the acyl-group on ghrelin, 1N hydrochloric acid was added to the p-hydroxymercuribenzoic acid-treated plasma to achieve a final concentration of 0.1 N. Processed samples were stored at −80 °C in small aliquots until further analysis of plasma hormone levels. ELISA kits were used for acyl-ghrelin (#EZRGRA-90K; Millipore-Merck, MA), GH (#EZRMGH-45K; Millipore-Merck), IGF-1 (#80574; Crystal Chem, IL), and LEAP2 [#EK-075-40; Phoenix Pharmaceuticals, Inc., CA]. Colorimetric assays were performed using a BioTek PowerWave XS Microplate spectrophotometer (BioTek, Winooski, VT) using BioTek KC4 junior software. Measurements of plasma AST, ALT, total cholesterol and triglycerides were measured with a VITROS analyzer (Ortho Clinical Diagnostics) in the UT Southwestern Mouse Metabolic Phenotyping Core.
8
Liver Tissue Analysis: Histology and Function
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Tissues were excised and fixed in 10% formalin solution and then stored in 50% ethanol. Embedding, processing, and staining of the tissue samples were performed by the UTSW Molecular Pathology Core or a company (Histoire, Seoul, Korea). For the liver function tests, albumin (ALB), alkaline phosphatase (AlkP), blood urea nitrogen (BUN), bilirubin, aspartate transaminase (AST), and alanine transaminase (ALT) were measured with a VITROS analyzer (Ortho Clinical Diagnostics) in the UTSW metabolic core. Anti-Ki67 (Diagnostic BioSystems, Pleasanton, CA), anti-alpha-SMA (Abcam, Cambridge, UK), anti-F4/80 (Sigma, St. Louis, MO), and anti-myeloperoxidase (Abcam) antibodies were used at a 1:200 dilution for immunohistostaining. The signals were detected with the Lab Vision™ UltraVision™ LP Detection System (Thermo Scientific, Waltham, MA). Apoptosis in paraffin-embedded liver sections was quantitated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) Kit (Merck, Burlington, MA) according to the manufacturer’s instructions.
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