Pancreatic islets were isolated by collagenase-P digestion (Roche Diagnostics, Indianapolis, IN) followed by centrifugation with Histopaque 1100 (Sigma-Aldrich, St. Louis, MO) as previously described (Carter, et al. 2009 (link)). Islets were incubated overnight in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin to allow recovery from collagenase digestion before further treatment. Various islet stressors were prepared as follows: Rotenone and thapsigargin were purchased from Sigma-Aldrich and prepared in stocks of DMSO to final concentrations less than 0.1%. Glucose-free RPMI medium (10% FBS and 1% penicillin/streptomycin) was supplemented with 1M glucose stock to produce the 28G condition. Stocks of the murine forms of IL-1B, IL-6, CXCL1, and CXCL5 (purchased from R&D Systems, Inc., Minneapolis, MN) were prepared in PBS with 0.1% BSA. Oleate, linOleate, and palmitate were purchased from Sigma-Aldrich and were prepared in 100mM stock concentrations in methanol and stored in -80. To treat the islets, the required amount of each fatty acid was taken in a glass tube and dried under a stream of nitrogen to remove methanol. The dried mixture of fatty acids thus obtained was resuspended in the RPMI medium with 0.1% BSA followed by vortexing and sonication. All experiments were completed within 48-72 hours post isolation.
Linoleate
Manufactured by Merck Group
Sourced in United States
Linoleate is a chemical compound that serves as a core component in various laboratory equipment and experiments. It functions as a building block for the production of essential materials used in scientific research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Oleate, by Merck Group (9 mentions)
Palmitate, by Merck Group (9 mentions)
Hek293t, by Merck Group (3 mentions)
Hepg2, by Merck Group (3 mentions)
Bovine serum albumin (bsa), by Merck Group (3 mentions)
Linolenate, by Merck Group (2 mentions)
Elaidate, by Merck Group (2 mentions)
Vaccenate, by Merck Group (2 mentions)
Palmitoleate, by Merck Group (2 mentions)
Collagenase p digestion, by Roche (1 mentions)
Cxcl5, by R&D Systems (1 mentions)
Histopaque 1100, by Merck Group (1 mentions)
Cxcl1, by R&D Systems (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Rotenone, by Merck Group (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Interleukin 6 (il 6), by R&D Systems (1 mentions)
A8806, by Merck Group (1 mentions)
D2141, by Merck Group (1 mentions)
D6429, by Merck Group (1 mentions)
11 protocols using linoleate
1
Pancreatic Islet Isolation and Treatment
2
Preparation of Fatty Acid-BSA Complexes
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The palmitate was prepared according to a previously described protocol (Cousin et al., 2001 (link)). Briefly, palmitate (#P0500; Sigma-Aldrich), oleate (#O1008; Sigma-Aldrich), linoleate (#L1012; Sigma-Aldrich), and linolenate (#L2376; Sigma-Aldrich) was solubilized in 0.1 M NaOH (heating at 70°C) at 100 mM, and combined with 10% (wt/vol) fatty acid–free BSA (#A8806; BSA, Sigma-Aldrich; heating at 55°C) to make a fatty acid concentration of 5 mM. Stearate (#S4751; Sigma-Aldrich) and arachidate (#A3631; Sigma-Aldrich) were solubilized in trichloromethane at 200 mM separately and combined with 10% (wt/vol) fatty acid–free BSA (heating at 55°C) to make a concentration of 2 mM. Stored fatty acid/10% BSA stock solutions were heated for 15 min at 55°C, and then cooled to room temperature before use.
3
Cholesterol Ester Isotope Synthesis
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Cholesteryl arachidonate, linoleate, oleate, palmitate, stearate, and octanoate-1- 13C1 were purchased from Sigma-Aldrich (St. Louis, MO), and stock solutions were prepared in dichloromethane at 1 mg/mL and stored under an atmosphere of N2 at -20°C (Table 2
4
Evaluating Gemcitabine Metabolism in Pancreatic Cancer Cells
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The K8484 cell line was maintained in DMEM (D6429, Sigma Aldrich Co Ltd.) containing 5% FBS (10270-098, Life Technologies Ltd., Paisley, UK). MIA PaCa-2 cells were obtained from the European Collection of Cell Cultures (Public Health England, Salisbury, UK, authenticated by STR genotyping) and maintained in DMEM containing 10% FBS. Gemcitabine (3259, Tocris Bioscience) was dissolved in DMSO; linoleate (L8134, Sigma Aldrich Co Ltd.) and 6-diazo-5-oxo-L-norleucine (DON, D2141 Sigma Aldrich Co Ltd.) were dissolved in sterile water. Experiments assessing the effect of DON on dFdC metabolism were carried out in media lacking glutamine (D5671, Sigma Aldrich Co Ltd.). Growth-inhibition assays were carried out using the sulphorhodamine assay, and subsequent synergy calculations were done as previously described (Lin et al, 2012 (link)).
5
Lipid Standards for Cell Culture
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Culture medium and supplements were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Oleate, palmitate, stearate, linOleate, elaidate, vaccenate, bovine serum albumin, HepG2, HEK293T, and SK-N-FI cells were purchased from Sigma-Aldrich (St. Louis, MO, USA). All of the chemicals that were used in this study were of analytical grade. All of the experiments and measurements were carried out by using Millipore ultrapure water.
6
Lipid Metabolism Analysis Protocol
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Culture medium and supplements were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Oleate, palmitate, palmitOleate, linOleate, stearate, cycloheximide, actinomycin D, HepG2, and HEK293T cells were purchased from Sigma-Aldrich (St. Louis, MO, USA). Polyclonal primary antibodies against SCD1 and Actin and secondary antibodies were obtained from Cell Signaling (Danvers, MA, USA). Antibody against Glu-Glu tag was purchased from Bethyl Laboratories (Montgomery, TX, USA). Methanol (gradient grade), isopropanol (gradient grade), n-hexane, and boron trifluoride-methanol solution were purchased from Merck (Rahway, NJ, USA). All other chemicals used in this study were of analytical grade. All experiments and measurements were carried out by using Millipore ultrapure water.
7
Evaluating Protein and mRNA Stability
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For protein stability assay, transfection medium was replaced after overnight incubation at 37 °C with 1 mL DMEM containing the translational inhibitor cycloheximide (50 μg/mL) and the cells were incubated for 1, 2, 4, or 6 h in 12-well plates. Non-transfected samples as well as cells transfected with pcDNA3.1(–) “empty” vector were used as control in all experiments.
For mRNA stability assay, transfection medium was replaced after overnight incubation at 37 °C with 1 mL DMEM containing the mRNA synthesis inhibitor actinomycin D (5 μg/mL) and the cells were incubated for 0, 1, 2, 4, 8, or 12 h in 12-well plates.
Oleate, palmitate, palmitOleate, linOleate, and stearate (Sigma-Aldrich, St. Louis, MO, USA) were diluted in ethanol (Molar Chemicals, Halásztelek, Hungary) to a final concentration of 50 mM, conjugated with 4.16 mM FA free BSA (Sigma-Aldrich, St. Louis, MO, USA) in 1:4 ratio, at 50 °C for 1 h. The working solution for FA treatments was prepared freshly in FBS-free and antibiotic-free medium at 100 µM final concentration. The culture medium had been replaced by FBS-free and antibiotic-free medium 1 h before the cells were treated with FA. The FA-treatment was carried out for 6 h in 12-well plates.
For mRNA stability assay, transfection medium was replaced after overnight incubation at 37 °C with 1 mL DMEM containing the mRNA synthesis inhibitor actinomycin D (5 μg/mL) and the cells were incubated for 0, 1, 2, 4, 8, or 12 h in 12-well plates.
Oleate, palmitate, palmitOleate, linOleate, and stearate (Sigma-Aldrich, St. Louis, MO, USA) were diluted in ethanol (Molar Chemicals, Halásztelek, Hungary) to a final concentration of 50 mM, conjugated with 4.16 mM FA free BSA (Sigma-Aldrich, St. Louis, MO, USA) in 1:4 ratio, at 50 °C for 1 h. The working solution for FA treatments was prepared freshly in FBS-free and antibiotic-free medium at 100 µM final concentration. The culture medium had been replaced by FBS-free and antibiotic-free medium 1 h before the cells were treated with FA. The FA-treatment was carried out for 6 h in 12-well plates.
8
Sterol and Fatty Acid Derivatives Synthesis
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Stigmasterol (≥95%), stearic acid (≥98.5%), oleic acid (≥99%), linoleic acid (≥99%), linolenic acid (≥99%), all solvents, sodium hydroxide, anhydrous pyridine, the catalysts-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP), high-purity silica gel 70–230 mesh, standards of cholesteryl stearate (96%), oleate (98%), linoleate (98%), linolenate (97%), 5α-cholestane and heptadecanoic acid methyl ester were purchased from Sigma-Aldrich (St. Louis, MO, USA). The silylation mixture Sylon BTZ was supplied by Fluka Analytical (Buchs, Switzerland). The internal standard 19-hydroxycholesterol was purchased from Steraloids (Newport, RI, USA). The silylation mixture of BSTFA [N,O-Bis(trimethylsilyl) trifluoroacetamide] with 1% TMCS (trimethylchlorosilane) was obtained from Fluka Chemie, while the SEP-PAK amino cartridges were sourced from Waters (Milford, USA).
9
Preparation of Fatty Acid-BSA Conjugates
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Palmitate, oleate, or linoleate (Sigma, St. Louis, MO) were dissolved in glass vials at 70 °C in a 50% Ethanol/Dulbecco’s phosphate buffered saline (DPBS) (Gibco, Thermo Fisher, Waltham, MA) solution and added to a solution of 10% fatty acid-free BSA (Sigma) that was pre-warmed to 55 °C. The fatty acid-BSA conjugates were mixed at 55 °C for 20 min then filter sterilized through 0.45 μm syringe filters (Millipore, Billerica, MA) and stored at − 20 °C in glass vials.
10
Fatty Acid Treatment Protocol
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FFA, palmitate and linoleate, were purchased from Sigma Chemical Company (St. Louis, MO). Each fatty acid was complexed with 5% bovine serum albumin and incubated with the cells at final concentrations of 100–1000 μM. FFA treatment was performed by palmitate (1 to 18 h, 100–800 μM) or linoleate alone (9 h, 100–1000 μM), or co-incubation with both of them (9 h, palmitate 500 μM and linoleate 100-1000 μM).
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