Following the dietary intervention and after an overnight fast (~12 hr), all animals were subjected to a metabolic study. In the morning of the study, animals were anesthetized with ketamine and xylazine, and fur shaved on the chest, neck and one ear. Jugular and marginal ear veins and the carotid artery were cannulated along with intubation of the trachea or placement of a laryngeal airway mask (LMA) and ventilated (Rate 12 bpm, Volume 100 mL pressure not to exceed 20 mmHg). Venous blood (~20 ml) in EDTA vacutainer tubes, and abdominal scAT and skeletal muscle samples from vastus lateralis were collected. Thereafter, a 3-hr primed continuous infusion of U-[13C16]-palmitate (99% enriched, Cambridge Isotope Laboratories, Inc., Tewksbury, MA) in 5% albumin (priming dose [PD]: 1.0 μmol•kg-1, infusion rate [IR]: 0.1 μmol•kg-1•min-1) was started (15 (link),16 (link)). About 3 ml of arterial blood was obtained at 30, 60, 90, 120, 150, 160, 170, and 180 min of infusion to determine the rate of appearance of palmitate [Ra] as a measure of the lipolysis rate. Thereafter, the animals were sacrificed by I.V. injection of 5 ml of Euthasol solution under general anesthesia of ketamine and xylazine. Death was confirmed by open chest observation. At this time, a liver sample was obtained.
U 13c16 palmitate
Manufactured by Cambridge Isotopes
U-[13C16]-palmitate is a stable isotope-labeled palmitic acid compound used in analytical and research applications. It contains a fully 13C-labeled palmitate molecule, which can be utilized as a tracer or internal standard in various analytical techniques.
Automatically generated - may contain errors
Lab products found in correlation
U 13c6 glucose, by Cambridge Isotopes (2 mentions)
U 13c5 glutamine, by Cambridge Isotopes (2 mentions)
Phenformin, by Merck Group (1 mentions)
Bptes, by Merck Group (1 mentions)
1 2 13c2 glucose, by Cambridge Isotopes (1 mentions)
Poly 2 hydroxyethyl methacrylate, by Merck Group (1 mentions)
Palmitic acid, by Merck Group (1 mentions)
Trace gc ultra, by Thermo Fisher Scientific (1 mentions)
Fumed silica, by Merck Group (1 mentions)
5 protocols using u 13c16 palmitate
1
Metabolic Study of Palmitate Kinetics
2
Stable Isotope Metabolic Tracing
3
Quantifying Cellular Metabolic Flux via GC/C/IRMS
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For 13C incorporation analysis, [U-13C6] glucose (1 mM; Cambridge Isotope Laboratories) or [U-13C16] palmitate (1 μM; Cambridge Isotope Laboratories) was added at the onset of the in vitro infection experiments. To determine the incorporation of glucose- or palmitate-derived carbon into cellular fatty acids, cells were saponified [MeOH:NaOH (15%) 1:1, 1 h, 100°C], derivatized [MeOH:HCl 10:2, 10 min, 80°C] and then prepared for analysis on a gas chromatography-combustion-isotope ratio mass spectrometer (GC/C/IRMS) as described earlier (49 (link)). GC/C/IRMS measurements were performed in triplicate on a Finnigan MAT 253 isotope ratio mass spectrometer coupled with a Trace GC Ultra (Thermo Fisher Scientific) chromatograph via a combustion interface. The fatty acid methyl esters were separated with an Optima five column (5% phenyl, 95% dimethylpolysiloxane, 50 m, 0.32 mm inner diameter, and 0.25 μm film thickness). The oven program was 100°C for 2 min, increased to 290°C at 4°C min−1, followed by an isothermal period of 8 min. The separated compounds were combusted on line in an oxidation oven. 13C/12C isotope ratios for the free fatty acids were calculated as described (49 (link)) and are presented as δ13C in the figures.
4
Metabolomics and Stable Isotope Tracing
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For metabolomics and stable isotope tracing, cells were cultured and differentiated in 6‐well plates using BAT differentiation media. On day 7 of differentiation cells were washed once and treated with 10 µM UK5099 or DMSO for 24 h in DMEM supplemented with phenol red, 10 mM glucose, 2 mM glutamine, and 10% NCS. For palmitate tracing experiments NCS was delipidated using fumed silica (Sigma‐Aldrich, St. Louis, MO), and 200 µM [U‐13C16] palmitate (Cambridge Isotope Laboratories, Tewksbury, MA) was added to the media, with unlabeled palmitate added to matched controls. Palmitate was added at a 4:1 palmitate:BSA complex. For glutamine‐tracing experiments, unlabeled glutamine in the media was replaced with 2 mM [U‐13C5] glutamine (Cambridge Isotope Laboratories, Tewksbury, MA). Metabolite extraction and GC/MS was performed as previously described in detail (Vacanti et al, 2014 ).
5
Fatty Acid Turnover Measurement
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