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9 10 3h palmitate

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[9,10-3H]palmitate is a radiolabeled fatty acid compound used in research and development applications. It serves as a tracer to study lipid metabolism and transport processes.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (3 mentions) Cytidine, by Merck Group (2 mentions) Guanosine, by Merck Group (2 mentions) Nucleoside mix, by Merck Group (2 mentions) Carnitine, by Merck Group (2 mentions) Methotrexate, by Merck Group (2 mentions) Oligomycin, by Merck Group (2 mentions) Dntp mix, by Merck Group (2 mentions) U 13c glucose, by Cambridge Isotopes (2 mentions) Hoechst 33342, by Thermo Fisher Scientific (2 mentions) U 13c glutamine, by Cambridge Isotopes (2 mentions) Purecol, by Advanced BioMatrix (2 mentions) U 13c acetate, by Cambridge Isotopes (2 mentions) L homopropargylglycine hpg, by Thermo Fisher Scientific (2 mentions) Uniformly labeled u 13c potassium palmitate, by Cambridge Isotopes (2 mentions) U 13c algal fatty acid mix, by Cambridge Isotopes (2 mentions) D 6 14c glucose, by PerkinElmer (2 mentions) 8 14c hypoxanthine, by PerkinElmer (2 mentions) U 14c palmitate, by PerkinElmer (2 mentions) L glutamine, by Thermo Fisher Scientific (2 mentions)

12 protocols using 9 10 3h palmitate

1

Measuring NEFA Uptake and β-Oxidation

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The rate of NEFA uptake and β-oxidation was measured by the intracellular accumulation of 9,10-[3H]-palmitate and the conversion of 9,10-[3H]-palmitate (Perkin Elmer) to [3H] labeled-H2O, respectively; using a modification of the method described previously (Supplementary Methods) [26] (link).
Armstrong M.J., Hull D., Guo K., Barton D., Hazlehurst J.M., Gathercole L.L., Nasiri M., Yu J., Gough S.C., Newsome P.N, & Tomlinson J.W. (2016). Glucagon-like peptide 1 decreases lipotoxicity in non-alcoholic steatohepatitis. Journal of Hepatology, 64(2), 399-408.
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2

Quantifying FAO Flux in Fibroblasts

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Flux through the FAO pathway was quantified by production of 3H2O from [9,10−3H] palmitate (PerkinElmer, Waltham, MA), conjugated to fatty acid-free albumin in fibroblasts cultured in a 24-well plate, as previously described (67 (link)). Palmitate bound to albumin was used at a final concentration of 12.4 μM (0.06 Ci/mmol). For each cell, FAO flux was measured in triplicate. The oxidation rates were expressed as pmol 3H-fatty acid oxidized/h/mg protein).
Seminotti B., Leipnitz G., Karunanidhi A., Kochersperger C., Roginskaya V.Y., Basu S., Wang Y., Wipf P., Van Houten B., Mohsen A.W, & Vockley J. (2018). Mitochondrial energetics is impaired in very long-chain acyl-CoA dehydrogenase deficiency and can be rescued by treatment with mitochondria-targeted electron scavengers. Human Molecular Genetics, 28(6), 928-941.
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3

Metabolic Labeling Techniques in Cell Culture

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Collagen type 1 (rat tail) was from Millipore or from PureCol® (bovine) (Advanced Biomatrix, USA). The CPT1a inhibitor (+)-etomoxir sodium salt hydrate was purchased from CNIO Carlos III Therapies. Mitomycin C (MitoC), sodium palmitate, dimethyl sulfoxide (DMSO), NAC, sodium acetate, oligomycin, cycloheximide, cytidine, adenine, guanosine, methotrexate, carnitine, dNTP mix and tamoxifen were from Sigma-Aldrich (Bornem, Belgium). 5-fluorouracil (TEVA Pharma Belgium) was obtained from the pharmacy of the university hospital Leuven. Nucleoside mix was from Millipore (Belgium)15 . Hoechst 33342 and L-homopropargylglycine (HPG) were from Molecular Probes and L-glutamine and penicillin/streptomycin were from Gibco® (Invitrogen, Life Technologies, Ghent, Belgium). Uniformly labeled [U-13C]-potassium palmitate, [U-13C]-acetate, [U-13C]-glucose, [U-13C]-glutamine and [U-13C]-algal fatty acid mix were obtained from Cambridge isotope laboratories, Inc. [U-14C]-palmitate, [9,10-3H]-palmitate, [6-14C]-D-glucose, [8-14C]-hypoxanthine and [3H]-thymidine were from Perkin Elmer.
Schoors S., Bruning U., Missiaen R., Queiroz K.C., Borgers G., Elia I., Zecchin A., Cantelmo A.R., Christen S., Goveia J., Heggermont W., Goddé L., Vinckier S., Van Veldhoven P.P., Eelen G., Schoonjans L., Gerhardt H., Dewerchin M., Baes M., De Bock K., Ghesquière B., Lunt S.Y., Fendt S.M, & Carmeliet P. (2015). Fatty acid carbon is essential for dNTP synthesis in endothelial cells. Nature, 520(7546), 192-197.
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4

Palmitate Labeling and Nmnat2 Transfection

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HEK 293 cells were maintained in culture, transfected and labeled with [9,10-3H]palmitate (PerkinElmer Life Sciences) as described (11 (link)). Where indicated, 50 μm PSB was added at the same time as the radiolabeled palmitate or 5 h after addition of palmitate (i.e. 1 h before cell lysis). HA (50 mm) was added for 60 min prior to cell lysis (i.e. 5 h after addition of radiolabel). Neuroblastoma X spinal cord cells (NSC34) were maintained in DMEM (Invitrogen) supplemented with 10% FBS (Sigma), 1% penicillin/streptomycin (Invitrogen), 2 mml-glutamine (Invitrogen), and 1 mm sodium pyruvate (Invitrogen). NSC34 cells in six-well dishes were transfected with 4 μg of FLAG-Nmnat2 and 1.35 μg of each of the indicated mouse siRNA construct(s) (Thermo Scientific) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Seventy-two hours after transfection, palmitate labeling was carried out as for HEK 293 cells.
Milde S, & Coleman M.P. (2014). Identification of Palmitoyltransferase and Thioesterase Enzymes That Control the Subcellular Localization of Axon Survival Factor Nicotinamide Mononucleotide Adenylyltransferase 2 (NMNAT2). The Journal of Biological Chemistry, 289(47), 32858-32870.
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5

Metabolic Labeling Techniques in Cell Culture

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Collagen type 1 (rat tail) was from Millipore or from PureCol® (bovine) (Advanced Biomatrix, USA). The CPT1a inhibitor (+)-etomoxir sodium salt hydrate was purchased from CNIO Carlos III Therapies. Mitomycin C (MitoC), sodium palmitate, dimethyl sulfoxide (DMSO), NAC, sodium acetate, oligomycin, cycloheximide, cytidine, adenine, guanosine, methotrexate, carnitine, dNTP mix and tamoxifen were from Sigma-Aldrich (Bornem, Belgium). 5-fluorouracil (TEVA Pharma Belgium) was obtained from the pharmacy of the university hospital Leuven. Nucleoside mix was from Millipore (Belgium)15 . Hoechst 33342 and L-homopropargylglycine (HPG) were from Molecular Probes and L-glutamine and penicillin/streptomycin were from Gibco® (Invitrogen, Life Technologies, Ghent, Belgium). Uniformly labeled [U-13C]-potassium palmitate, [U-13C]-acetate, [U-13C]-glucose, [U-13C]-glutamine and [U-13C]-algal fatty acid mix were obtained from Cambridge isotope laboratories, Inc. [U-14C]-palmitate, [9,10-3H]-palmitate, [6-14C]-D-glucose, [8-14C]-hypoxanthine and [3H]-thymidine were from Perkin Elmer.
Schoors S., Bruning U., Missiaen R., Queiroz K.C., Borgers G., Elia I., Zecchin A., Cantelmo A.R., Christen S., Goveia J., Heggermont W., Goddé L., Vinckier S., Van Veldhoven P.P., Eelen G., Schoonjans L., Gerhardt H., Dewerchin M., Baes M., De Bock K., Ghesquière B., Lunt S.Y., Fendt S.M, & Carmeliet P. (2015). Fatty acid carbon is essential for dNTP synthesis in endothelial cells. Nature, 520(7546), 192-197.
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6

Palmitate Uptake and Oxidation Assay

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HAOEC were submitted to shear stress or left under static condition in complete MV2 medium with 100 μM unlabeled palmitate and 50 μM carnitine. Cells were then incubated for 2 h in complete medium containing 2 μCi/mL [9,10 3H] palmitate (Perkin Elmer, France). Medium was transferred into glass vial and [3H]-H2O was captured on a piece of Whatman paper soaked with H2O over a period of 48 h at 37°C. Radioactivity was measured by liquid scintillation counting (Schoors et al., 2015 (link); Kalucka et al., 2018 (link)).
Bousquet D., Nader E., Connes P, & Guillot N. (2024). Liver X receptor agonist upregulates LPCAT3 in human aortic endothelial cells. Frontiers in Physiology, 15, 1388404.
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7

Measuring Cellular Metabolic Fluxes

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50,000 cells per well were seeded and allowed to attach before gentle washing and incubation for 48 hours in media containing 0.4 μCi/mL (24.24 nM final concentration) [5-3H]glucose (Perkin Elmer) or 2 μCi/mL (66.67 nM final concentration) [9,10-3H]palmitate (Perkin Elmer) for assessment of glycolytic or FAO flux, respectively. For FAO flux assays, 50 μM L-carnitine solution (MilliporeSigma) was supplemented to the media. To assess the production of 3H2O after a cumulative 48 hours of drug treatment, media was transferred to glass vials and sealed with rubber stoppers. 3H2O was captured in hanging wells within the glass vials containing Whatman filter paper soaked with equivolume H2O over a period of 48 hours at 37°C to reach saturation. Radioactivity for radioisotope tracer studies was determined by liquid scintillation counting using a Beckman Coulter LS6500 scintillation counter.
Zhu Y., Dun H., Ye L., Terada Y., Shriver L.P., Patti G.J., Kreisel D., Gelman A.E, & Wong B.W. (2022). Targeting fatty acid β-oxidation impairs monocyte differentiation and prolongs heart allograft survival. JCI Insight, 7(7), e151596.
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8

Quantifying Palmitate Oxidation in Fibroblasts

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FAO flux quantifies the rate of [9,10-3H] Palmitate (PerkinElmer, Waltham, MA) oxidation. [9,10-3H] Palmitate is emulsified in Krebs/BSA (4.5 mg/ml) solution to a final concentration of 23 μM overnight at 37 °C under continuous agitation. The fibroblasts were seeded in 24-well plates (1 × 105 cell/well) and were incubated for 4 h with 200 μl of the [9,10-3H] palmitate emulsified Krebs/BSA solution. The 3H2O released in the cell media was quantified at the end of the 4-h incubation. Etomoxir (2[6(4-chlorophenoxy) hexyl]oxirane-2-carboxylate, 10 μM) an irreversible inhibitor of carnitine palmitoyltransferase-1 (CPT-1α) was used to document mitochondrial-dependent β-oxidation of palmitate. The assay was performed in three different cell preparations for each cell type using cells of similar passage (p4-p10). For each independent determination, non-disease and fibroblasts with mutations were analyzed in duplicate. For this assay we used control, non-disease fibroblast lines 1 and 2. The flux is expressed in pmol/h/mg and the data is reported as the percent of the control 1 fibroblasts.
Raimo S., Zura-Miller G., Fezelinia H., Spruce L.A., Zakopoulos I., Mohsen A.W., Vockley J, & Ischiropoulos H. (2021). Mitochondrial morphology, bioenergetics and proteomic responses in fatty acid oxidation disorders. Redox Biology, 41, 101923.
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9

Measuring Fatty Acid and Glucose Oxidation in Osteoclasts

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FA and glucose oxidation were measured in osteoclasts derived from RAW 264.7 cells after 5 days of treatment with RANKL and undifferentiated (day 1 post-RANKL stimulation) RAW 264.7 cells. Cells were cultured as described above. On day 1 and day 5, cells were harvested in PBS, incubated at 37°C for 2 hours in modified Krebs-Ringer buffer (MKR) (115 mm NaCl, 2.6 mm KCl, 1.2 mm KH2PO4, 10 mm NaHCO3, 10 mm HEPES [pH 7.4]) that contained 2% BSA, 0.2 mmol/mL palmitate, and 10 μCi/mL [9,10-3H]palmitate (PerkinElmer, Waltham, MA, USA) for assessment of FA oxidation or MKR that contained 0.2 mmol/mL glucose and 10 μCi/ml D-[3-3H]glucose (PerkinElmer) for assessment of glucose oxidation and were gassed with 95% O2 and 5% CO2. Then, water was extracted with chloroform: methanol (2:1) extraction. palmitate or glucose oxidation was determined by measuring the amount of 3H2O in the aqueous phase.
Drosatos-Tampakaki Z., Drosatos K., Siegelin Y., Gong S., Khan S., Van Dyke T., Goldberg I.J., Schulze P.C, & Schulze-Späte U. (2014). Palmitic Acid and DGAT1 Deficiency Enhance Osteoclastogenesis, while Oleic Acid-Induced Triglyceride Formation Prevents It. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, 29(5), 1183-1195.
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10

Measuring Fatty Acid Oxidation in Thymic Lymphocytes

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Fatty acid oxidation was measured using 3H-palmitate as previously described (19 (link)). Briefly, 2 × 106 freshly isolated thymic lymphocytes were resuspended in 1 mL of modified KHB medium (111 mM NaCl, 4.7 mM KCl, 1.25 mM CaCl2, 2 mM MgSO4, 1.2 mM NaH2PO4, 2.5 mM glucose, 0.5 mM carnitine, 5 mM HEPES, pH 7.4) with or without 200 μM etomoxir. A mixture of [9,10-3H] palmitate (53.7 Ci/mmol, PerkinElmer, Boston, MA) and 10% BSA in KHB buffer was added to the cell suspension and incubated at 37 °C for 2.5 h. After incubation, the cells were spun down at 300x g and assayed for protein content while the supernatant was subjected to chloroform-methanol extraction to measure the 3H2O product in the aqueous phase. The etomoxir-sensitive 3H2O generated was used to calculate the β-oxidation rate and expressed as CPM/μg protein.
Wang P.Y., Ma J., Li J., Starost M.F., Wolfgang M.J., Singh K., Pirooznia M., Kang J.G, & Hwang P.M. (2020). Reducing fatty acid oxidation improves cancer-free survival in a mouse model of Li-Fraumeni syndrome. Cancer prevention research (Philadelphia, Pa.), 14(1), 31-40.
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