Mice body composition was assessed by non-invasive MRI scanning on an EchoMRI 700 (BUSM Metabolic Phenotyping core). Glucose Tolerance Test (GTT) and Insulin Tolerance Test (ITT) were performed according to established protocols [44] (link). Briefly, mice were starved overnight or 4–6 h for GTT and ITT, respectively. Blood glucose levels were measured, at the described time points after glucose (1.5 mg/g body weight) or insulin (Humulin R, Lilly) (0.5 U/kg body weight) IP injection, from tail nicking using a OneTouch Ultra glucometer. Plasma was collected from overnight fasted mice after cardiac puncture and profiled using Milliplex Multiplex Assays (Millipore, BUSM Analytical Core) and Free Fatty Acid Fluorometric Assay (Cayman Chemical).
Free fatty acid fluorometric assay
Manufactured by Cayman Chemical
Sourced in United States
The Free Fatty Acid Fluorometric Assay is a laboratory tool used to measure the concentration of free fatty acids in a sample. It provides a quantitative analysis of free fatty acid levels without interpretation or extrapolation of the intended use.
Automatically generated - may contain errors
Lab products found in correlation
Milliplex multiplex assay, by Merck Group (1 mentions)
Humulin r, by Eli Lilly (1 mentions)
Glycerol assay kit, by Merck Group (1 mentions)
Mouse tnf alpha quantikine hs elisa kit, by R&D Systems (1 mentions)
Enzychrom ketone body assay kit, by BioAssay Systems (1 mentions)
Epoc blood analysis system, by Siemens (1 mentions)
Novex see blue plus 2, by Thermo Fisher Scientific (1 mentions)
Gel logic 2200 imaging system, by Kodak (1 mentions)
3 protocols using free fatty acid fluorometric assay
1
Metabolic Phenotyping of Mice
2
Metabolic Biomarkers in Sepsis Survival
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For surviving animals, after 5 days of illness, blood pH and blood Na+, K+, Cl−, HCO3−, and creatinine concentrations were measured at sacrifice with use of the Epoc® Blood Analysis System (Siemens Healthineers, The Hague, The Netherlands). Whole blood 3HB− concentrations were measured with the StatStrip Xpress®2 Glucose/Ketone meter (Nova Biomedical, Waltham, MA, USA) 30 min after injection of the study dose on day 1. In plasma collected at sacrifice, 3HB− was quantified with a commercial enzymatic kit (EnzyChrom™ ketone body assay kit, Bioassay Systems, Hayward, CA, USA). Impact of 3HB-Na on inflammation was assessed by quantification of plasma TNFα (Mouse TNF-alpha Quantikine HS ELISA Kit, R&D systems, Minneapolis, MN, USA). As ketone bodies can suppress lipolysis which may have detrimental effects during sepsis [10 (link), 19 (link)], plasma free fatty acids (Free Fatty Acid Fluorometric Assay, Cayman, Ann Arbor, MI, USA) and glycerol (Glycerol Assay Kit, Sigma-Aldrich) were quantified. Plasma aldosterone (All species Aldosterone ELISA Kit, LSBio, Seattle, WA, USA) was quantified as a marker of the renin-angiotensin-aldosterone system, which is involved in fluid retention and can be affected by salt intake.
3
SDS-PAGE and Fatty Acid Analysis
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Sodium dodecyl sulfate-polyacrylamide gel-electrophoresis (SDS-PAGE) was carried out as previously described 22 to study the extent of protein digestion. Samples were denatured at 95 o C for 5 min. and were then loaded to a 15% acrylamide and 0.4% bisacrylamide gel for the samples obtained after complete GI digestion, or to a 10% acrylamide/0.3% bisacrylamide gel for the digesta obtained following only gastric digestion. The running time was 30 min. at 80 V followed by 90 min. at 100 V. Protein marker was Invitrogen novex see blue plus 2 (Invitrogen, Carlsbad, CA). After each run, fixation/staining was done with methanol/acetic acid/Coomassie brilliant blue R (50%, 10%, 0.1%) for 30 minutes, and destained in methanol/glacial acetic acid/water (30%, 10%, 60%, 3 h). The gels were scanned by a Kodak Gel Logic 2200 imaging system (Kodak, Rochester, NY).
Free fatty acid determination: Lipid hydrolysis was evaluated by measuring the amount of free fatty acids (FFAs) released in the digesta after GI digestion. This was determined by Cayman's Free Fatty Acid Fluorometric Assay (Cayman Chemical, Art. No. 700310, Ann Arbor, MI) according to the manufacturer's protocol.
Free fatty acid determination: Lipid hydrolysis was evaluated by measuring the amount of free fatty acids (FFAs) released in the digesta after GI digestion. This was determined by Cayman's Free Fatty Acid Fluorometric Assay (Cayman Chemical, Art. No. 700310, Ann Arbor, MI) according to the manufacturer's protocol.
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