Infinity Cholesterol (catalog #TR13421), Infinity Triglyceride (catalog #TR22421), and TRIzolTM (catalog #15596018) reagents were purchased from Thermo Fisher Scientific (Middletown, VA, USA). Autokit Glucose (catalog #997-03001), Phospholipids C (catalog #997-01801), and HR Series NEFA-HR(2) (catalog #999-34691, 995-34791, 991-34891, and 993-35191) kits were purchased from Fujifilm Wako Chemicals USA (Richmond, VA, USA). Omniscript RT (catalog #205113) kit was purchased from Qiagen (Germantown, MD, USA) and qPCRTM core kit for SYBR Green I (catalog #10-SN10-05) was from Eurogentec (San Diego, CA, USA). 3H-cholesterol (catalog #NET139001MC), 14C-oleic acid (catalog #NEC317250UC), and 14C-triolein (catalog #NEC674250UC) were from PerkinElmer (Shelton, CT, USA). Poloxamer 407 (catalog #P1166) was purchased from Spectrum Chemical (New Brunswick, NJ, USA). Primary and secondary antibodies were purchased from either Cell Signaling (Danvers, MA, USA) or Abcam (Cambridge, MA, USA). All other chemicals and solvents were obtained from Fisher Scientific through its local distributor in the Kingdom of Saudi Arabia.
14c oleic acid
Manufactured by PerkinElmer
Sourced in United States
14C-oleic acid is a radioactively labeled lipid compound. It is used as a research tool to study lipid metabolism and transport processes in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Oleic acid, by Merck Group (2 mentions)
Ultima gold scintillation cocktail, by Merck Group (2 mentions)
Bovine serum albumin (bsa), by Merck Group (2 mentions)
Infinity triglyceride, by Thermo Fisher Scientific (1 mentions)
Autokit glucose, by Fujifilm (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Hr series nefa hr 2, by Fujifilm (1 mentions)
Qpcr core kit for sybr green 1, by Eurogentec (1 mentions)
Infinity cholesterol, by Thermo Fisher Scientific (1 mentions)
Phospholipids c, by Fujifilm (1 mentions)
14c triolein, by PerkinElmer (1 mentions)
3h cholesterol, by PerkinElmer (1 mentions)
Omniscript rt, by Qiagen (1 mentions)
Poloxamer 407, by Spectrum Chemical (1 mentions)
3 isobutyl 1 methylxanthine, by Merck Group (1 mentions)
Anti flag m2 agarose, by Merck Group (1 mentions)
Fugene hd, by Promega (1 mentions)
Protein g agarose, by Thermo Fisher Scientific (1 mentions)
Anti ubiquitin antibody, by Santa Cruz Biotechnology (1 mentions)
Dexamethasone (dex), by Merck Group (1 mentions)
15 protocols using 14c oleic acid
1
Comprehensive Lipid Metabolism Profiling
2
Lipid Transport and Fatty Acid Oxidation in Amygdala Neurons
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In vitro lipid transport assay. Primary amygdala neurons were seeded in a 12-well plate and grew until they were 80% confluent. After treatment, 0.5 mCi well−1 of 14C oleic acid (OA) from PerkinElmer was added. After 4 h of incubation, the cells were washed and harvested, and the total radioactivity was quantitated by scintillation counting [34 (link)].
Rate of fatty acid oxidation from tissues. The fatty acid oxidation (FAO) rate was measured by evaluation of palmitate oxidation using published methods with minor modifications [35 (link), 36 (link)]. In brief, the amygdala tissue was homogenized, and 30 μl of tissue homogenate were then incubated in 370 μl of DMEM containing 0.5% BSA/0.2 mM palmitate/0.5 μCi/ml 1-14C-palmitate at 37 °C for 2 h. The incubation was stopped by the injection of 0.2 ml of 40% perchloric acid into the tube to acidify the medium and liberate the CO2. The CO2 was trapped by a filter paper saturated with 20 μL of 1 M NaOH located on the top of the cap. After overnight isotopic equilibration at room temperature, the filter was removed, and the trapped 14CO2 and 14C acid-soluble products generated by the oxidation of [14C] palmitate were counted to calculate the total palmitate oxidation. The protein concentrations were measured, and the results were expressed as nanomole per milligram proteins per hour (nmol/mg/h).
Rate of fatty acid oxidation from tissues. The fatty acid oxidation (FAO) rate was measured by evaluation of palmitate oxidation using published methods with minor modifications [35 (link), 36 (link)]. In brief, the amygdala tissue was homogenized, and 30 μl of tissue homogenate were then incubated in 370 μl of DMEM containing 0.5% BSA/0.2 mM palmitate/0.5 μCi/ml 1-14C-palmitate at 37 °C for 2 h. The incubation was stopped by the injection of 0.2 ml of 40% perchloric acid into the tube to acidify the medium and liberate the CO2. The CO2 was trapped by a filter paper saturated with 20 μL of 1 M NaOH located on the top of the cap. After overnight isotopic equilibration at room temperature, the filter was removed, and the trapped 14CO2 and 14C acid-soluble products generated by the oxidation of [14C] palmitate were counted to calculate the total palmitate oxidation. The protein concentrations were measured, and the results were expressed as nanomole per milligram proteins per hour (nmol/mg/h).
3
Characterization of LDAH Antibodies
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Custom polyclonal rabbit anti-human and mouse LDAH antibodies were generated at Bethyl Laboratories, and characterized as previously described15 (link). Bodipy 493/503, LipidTox, and RNAiMAX were from Invitrogen. Fugene HD was purchased from Promega. Isoproterenol hydrochloride, anti-beta actin antibody, anti Flag-M2-HRP, anti Flag-M2-agarose, mouse IgG, oleic acid, 3-isobutyl-1-methylxanthine, insulin, dexamethasone and nocodazole were from Sigma-Aldrich. Triacsin C and anti-ubiquitin antibody were from Santa Cruz Biotechnology. Anti-PLIN1 antibody was from Progen. Anti-beta tubulin and anti-PLIN2 antibodies were from Nobus Biologicals. Anti-PLIN3 was purchased from Fitzgerald. Anti-ATGL and anti-HSL were from Cell Signaling. Alexa Fluor 647-conjugated secondary antibodies and anti-GM130 were from BD Pharmingen. Protein G-agarose and protein-A-agarose were from Thermo Scientific. MG132 was purchased from Calbiochem. Total and free cholesterol were measured using kits from Wako. TAG were determined with Infinity Trglyceride reagents from Thermo Scientific. 14C-oleic acid was purchased from Perkin Elmer.
4
Antioxidant Assays with Viper Venom
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Sinapic acid, gallic acid (GA), thiobarbituric acid (TBA), Sephadex (G25, G50, and G75), CM-Sephadex-25, 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH), Ultima Gold Scintillation Cocktail, and dimethyl sulfoxide (DMSO) areproducts of Sigma-Aldrich, St. Louis, MO, USA. 14C-oleic acid was procured from Perkin Elmer Life Sciences Inc. in Boston, MA, USA. The venom of Viper russelli was purchased from Irula Cooperative Society Ltd., Tamil Nadu, India. All reagents and chemicals used in the investigation were of superior quality.
5
Fatty Acid Oxidation in VAD Implantation
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Fatty acid oxidation in muscle tissues was performed in rectus abdominis muscle samples obtained from patients before and after VAD implantation (n = 6 per group) by measuring the production of [14C] CO2 from [14C] oleic acid (PerkinElmer, Waltham, MA). Approximately 20–30 mg of muscle tissues was transferred into 10-mL Kontes® flasks (Sigma-Aldrich, St. Louis, MO) containing 1 mL of modified Krebs Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 1.2 mM KH2PO4, 10 mM NaHCO3, 10 mM HEPES) with 1.5 % BSA, 0.2 mM oleic acid, 2 μCi/mL [14C] oleic acid, and 1 mM glucose. The flasks were immediately cupped with rubber stoppers containing an attached central well with a 3 × 3 cm filter paper soaked in 300 μL KOH. After 1 h of incubation at 37 °C, 200 μL of 70 % perchloric acid was injected into the flask. The flasks were incubated for an additional 1 h at 37 °C with shaking at 70 Hz. The filter papers containing the trapped [14C] CO2 were transferred to liquid scintillation tubes and assessed for radioactivity in a liquid scintillation counter.
6
Biochemical Characterization of Vipera russelii Venom
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Procured elemolic acid, gallic acid, 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH˙), thiobarbituric acid (TBA), Sephadex (G-25, 50 and 75), CM-Sephadex C-25, Scintillation cocktail (Ultima Gold) and dimethyl sulfoxide (DMSO) from Sigma-Aldrich (St. Louis, MO, USA). 14C-oleic acid was purchased from Perkin Elmer Life Sciences Inc. Boston, USA. The Vipera russelii venom was obtained from Irula Cooperative Society Ltd., Chennai, India. All other chemicals and reagents used in this study were in higher quality.
7
Biochemical Analysis of Biological Fluids
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14C-oleic acid was obtained from PerkinElmer Life Sciences Inc., Boston, MA, USA. Fatty acid-free bovine serum albumin (BSA) fraction V was purchased from PAA Laboratories, GmbH Haidmannweg, Austria. Agar, beef extract, peptone, and lactose were obtained from HiMedia Laboratories Private Limited, Mumbai, India. Scintillation cocktail (Ultima Gold) was obtained from Packard Bioscience, USA. Escherichia coli (lyophilized cells of strain W [ATCC 9637]), Sephadex (G-25, 50, and 75), CM-Sephadex C-25, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙), thiobarbituric acid (TBA), gallic acid (GA), dimethyl sulfoxide (DMSO), oleanolic acid were purchased from Sigma-Aldrich Chemical Laboratories, St. Louis, MO, USA. Human pleural fluid (HPF) and synovial fluid were obtained from Princes Krishnajammanni Tuberculosis and Chest Disease Hospital, Mysore, and Dr. Hegde Orthopaedic Clinic, Mysore, India. Blood samples for indirect hemolytic activity were obtained from the healthy volunteers, Department of Studies in Biochemistry, University of Mysore. All other chemicals and reagents used in this study were of analytical grade or better and solvents were redistilled before use.
8
Characterization of Anti-inflammatory Compounds
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Sephadex G-25, −50 and −75, CM sephadex C25 Corosolic acid, Oleanolic acid and Lipopolysaccharide (#L2018), Ultima Gold Scintillation Cocktail, Bovine serum albumin (BSA), were procured from Sigma-Aldrich, USA.14C-oleic acid obtained from Perkin Elmer Life Sciences Inc. Boston. Mouse Anti-Human IL-6 (Cat No: 340527) was from B.D Biosciences. Six-well cell culture plates from BioLite - Thermo. The PC3 Cell line was obtained from the national centre for cell science (NCCS), Pune, the cell culture medium (RPMI 1640, #AL028A), Fetal Bovine Serum (#RM10432) and dulbecco’s phosphate buffered saline (D-PBS) (#TL1006) were procured from HiMedia Laboratories, Mumbai, India. Ethylenediaminetetraacetic acid (EDTA) and dimethyl sulphoxide (DMSO) were acquired from SRL (Sisco Research Laboratories), India. The solvents and chemicals utilised in the entire investigation were standard laboratory grade.
9
Biochemical Characterization of Recombinant Caspase-4
10
Oleic Acid Uptake and Incorporation
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Cells were incubated in the presence of 5.5 mM glucose and 0.5% (w/v) BSA-complexed with 0.375 mM oleic acid and 0.1 μCi [14C]-oleic acid (PerkinElmer) for 24 h. Cells were collected in 1 ml 2% NaCl–PBS buffer. Lipids were extracted by adding 2 ml of methanol and 1 ml chloroform and centrifuged (2500 rev/min for 10 min at room temperature). H2O (1 ml) and chloroform (1 ml) were added and again centrifuged (2500 rev/min, for 10 min at room temperature). The upper phase was removed and the lower phase dried under nitrogen and solubilized in 100 μl of chloroform and applied on a TLC-plate. The TLC-plates were run in a chamber containing (N-hexane, diethylether, acetic acid and H2O (65/15/1/0.25, v/v). Iodine vapour stained areas containing the TAGs were scraped out from the TLC-plate and radioactivity was measured by liquid scintillation counting (Wallac LS-Beta-Counter).
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