14c cholesterol

Manufactured by PerkinElmer

Sourced in United States

14C-cholesterol is a radiolabeled compound used in research applications. It contains the carbon-14 isotope, allowing it to be tracked and detected through various analytical techniques. The core function of 14C-cholesterol is to serve as a tracer molecule for studying cholesterol metabolism and related biological processes.

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[4-14C]-cholesterol (6 citations) [1-14C]-cholesterol (2 citations)

7 protocols using 14c cholesterol

Cholesterol Efflux Assay for Hepatocytes

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Lipid efflux assays were performed as previously described (Lyssenko et al., 2013 (link)). Twenty days after differentiation, HLCs were radiolabeled, with 0.12 μCi/ml ¹⁴C-cholesterol and 1.3 μCi/ml ³H-choline or with 0.12 μCi/ml ¹⁴C-cholesterol only (PerkinElmer, Waltham, MA, USA) in the presence of T0901317 (10 μM, Sigma-Aldrich T2320) for 24 h. Cells were then washed twice and incubated in fresh medium with or without exogenous human apoA-I (20 μg/ml) for 6 h. At the end of incubation, cell medium was collected and filtered through a 96-well filter plate (EMD Millipore) to remove cell debris. ApoB-containing lipoprotein was precipitated using phosphotungstate. Remaining HDL-containing medium underwent lipid extraction by Bligh-Dyer method. Cellular lipids were extracted with hexane-isopropanol (3:2, v/v) and the solvent was then evaporated for scintillation counting. The percentage of cholesterol export was calculated by dividing the ¹⁴C counts in the medium by the sum of counts in the medium and cells and multiplying by 100.

Bi X., Pashos E.E., Cuchel M., Lyssenko N.N., Hernandez M., Picataggi A., McParland J., Yang W., Liu Y., Yan R., Yu C., DerOhannessian S.L., Phillips M.C., Morrisey E.E., Duncan S.A, & Rader D.J. (2017). ATP-Binding Cassette Transporter A1 Deficiency in Human Induced Pluripotent Stem Cell-Derived Hepatocytes Abrogates HDL Biogenesis and Enhances Triglyceride Secretion. EBioMedicine, 18, 139-145.

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Cholesterol Metabolism and Regulation Analysis

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Deuterated sterols were purchased from Medical Isotopes Inc. Radiolabeled sterols, including [³H]cholesterol, [³H]cholesteryl oleate, and [¹⁴C]cholesterol, were purchased from PerkinElmer Inc. Bile acid levels and phospholipids were measured using kits from Crystal Chem Inc. and Wako Chemicals USA Inc., respectively. Abs against mouse G5 and G8 were developed as previously described (11 (link)); Abs against ABCA1 were purchased from Pierce Biotechnology Inc. All other Abs, including polyclonal Abs against low density lipoprotein receptor (LDLR) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) and monoclonal Abs against sterol-regulatory element binding protein (SREBP) 1 and SREBP-2 were gifts from Guosheng Liang (University of Texas Southwestern Medical Center).

Wang J., Mitsche M.A., Lütjohann D., Cohen J.C., Xie X.S, & Hobbs H.H. (2015). Relative roles of ABCG5/ABCG8 in liver and intestine. Journal of Lipid Research, 56(2), 319-330.

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Bryonolic Acid Purification and Characterization

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Bryonolic acid (BrA) was purified and characterized exactly as described earlier [5 (link)]. Betulinic and ursolic acids were purchased from Extrasynthese (Lyon Nord, Genay, France). Acetic acid, acetonitrile, dichloromethane (DCM) and anhydrous sodium sulphate were obtained from Merck (Darmstadt, Germany). 3-[decyldimethylsilyl]-N-[2-(4-methylphenyl)-1-phenylethyl]-propanamide (Sah 58-035) was kindly provided by A. Suter at Novartis (Basel, Switzerland). [³H]17βEstradiol, [¹⁴C]oleyl-CoA and [¹⁴C] cholesterol were from Perkin Elmer (Waltham, MA, USA). The radiochemical purity of the compounds was verified by thin-layer chromatography (TLC) and was greater than 98%. Solvents were from Sigma, Fischer, Scharlau or VWR. TLC plates were from Macherey Nagel. Normal silica Sep-Pack cartridges were from Waters. All other chemicals and reagents were purchased from Sigma-Aldrich (Saint-Louis, MO, USA).

Khallouki F., Owen R.W., Silvente-Poirot S, & Poirot M. (2018). Bryonolic Acid Blocks Cancer Cell Clonogenicity and Invasiveness through the Inhibition of Fatty Acid: Cholesteryl Ester Formation. Biomedicines, 6(1), 21.

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Cholesterol Efflux Assay with ApoA-IV

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BMDMs were loaded with cholesterol by incubation for 48 h with acetylated LDL (50 μg of protein/mL) and ¹⁴C-cholesterol (0.3 uCi/mL, Perkin Elmer, Boston, MA) then incubated with DMEM containing fatty acid-free bovine serum albumin (BSA) for 24 h to equilibrate cell cholesterol pools. The cells were then incubated for 8 h with unmodified-apoA-IV or AGE-apoA-IV (30 μg/mL). Radioactivity was measured in the medium and in the cells. The % cholesterol efflux was calculated as described by Machado et al. [47 (link)].

Shimabukuro Okuda L., Tallada Iborra R., Ramos Pinto P., Fabres Machado U., Corrêa-Giannella M.L., Pickford R., Woods T., Brimble M.A., Rye K.A, & Passarelli M. (2020). Advanced Glycated apoA-IV Loses Its Ability to Prevent the LPS-Induced Reduction in Cholesterol Efflux-Related Gene Expression in Macrophages. Mediators of Inflammation, 2020, 6515401.

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Cholesterol Metabolism Reagents Protocol

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The following chemicals were purchased from Sigma: angiotensin II (cat # A9525), dexamethasone (cat # D4902), dibutyryl cyclic-AMP sodium salt (cat # D0627), forskolin (cat # F6886), glucagon (cat # G2044), hydrocortisone (cat # H0888), InSolution H-89 dihydrochloride (cat # 371962), PKA Inhibitor 14-22 Amide (cat # 476485), and tyloxapol (cat # T0307). ¹⁴C-cholesterol (cat # NEC018050UC), ³H-cholesterol (cat # NET139001MC), ³H-cholesteryl oleate (cat # NET746L001MC) and ³H-triolein (cat # NET431001MC) were purchased from PerkinElmer.

Xu Y., Li Y., Jadhav K., Pan X., Zhu Y., Hu S., Chen S., Chen L., Tang Y., Wang H.H., Yang L., Wang D.Q., Yin L, & Zhang Y. (2021). Hepatocyte ATF3 protects against atherosclerosis by regulating HDL and bile acid metabolism. Nature metabolism, 3(1), 59-74.

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Nascent HDL Formation and Gel-Filtration

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Nascent HDL formation and gel-filtration chromatography analysis of nascent HDL were performed as previously described (Lyssenko et al., 2013 (link)). Briefly, HLCs were dual radiolabeled with 1.3 μCi/ml ³H-choline and 0.12 μCi/ml ¹⁴C-cholesterol (PerkinElmer, Waltham, MA, USA) in the presence of 10 μM T0901317 (Sigma-Aldrich T2320) for 24 h. After two washes, efflux medium with or without human apoAI (20 μg/ml) were added to cells for a 6-hour incubation. Cell medium was collected, filtered through a 0.45 μm PVDF membrane filter unit (EMD Millipore), and concentrated using Amicon Ultracel-10 K centrifugal filters (EMD Millipore). A 1-ml aliquot of the concentrated cell medium was resolved into 1-ml fractions on a calibrated HiLoad 16/60 Superdex 200 gel-filtration column (GE Healthcare, Mickleton, NJ, USA). ³H and ¹⁴C counts in each fraction were determined by scintillation counting. Results were presented as counts per fraction for regions containing HDLs (fraction 45–90).

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Cholesterol Metabolism Reagents Protocol

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