Cholesterol, noncholesterol sterol, and oxysterol content was analyzed in macrophages and Jurkat cells by gas chromatography (GC). Cell pellets were spun in a speedvac concentrator (12 mbar; Savant AES 1000) and weighed. Cholesterol, noncholesterol sterols, and oxysterols were extracted using chloroform. After alkaline hydrolysis, the concentrations of cholesterol precursors were measured with GC-mass spectrometry-selected ion monitoring [19 (link)]. The trimethylsilyethers of the sterols were separated on a DB-XLB (30 m length × 0.25 mm internal diameter, 0.25 μm film) column (Agilent Technologies, Waldbronn, Germany) using the 6890N Network GC system (Agilent Technologies). Epicoprostanol (Steraloids, Newport, RI, USA) was used as an internal standard, to quantify the noncholesterol sterols (Medical Isotopes, Pelham, NH, USA) on a 5973 Network MSD (Agilent Technologies). Total cholesterol was measured by GC-flame ionization detection on an HP 6890 GC system (Hewlett Packard, Waldbronn, Germany), equipped with a DB-XLB (30 m length × 0.25 mm internal diameter, 0.25 μm film) column (Agilent Technologies) using 5a-cholestane (Steraloids) as internal standard [20 ].
Epicoprostanol
Manufactured by Steraloids
Sourced in United States
Epicoprostanol is a chemical compound used in laboratory settings. It functions as a chemical intermediate in various research and analytical processes.
Automatically generated - may contain errors
2 protocols using epicoprostanol
1
Quantifying Sterols in Macrophages and Jurkat Cells
2
Quantification of Cellular Sterols and Oxysterols
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The cellular content of cholesterol was measured by gas chromatography-flame ionization detection (GC-FID) and of non-cholesterol sterols and oxysterols by gas chromatography-mass spectrometry in selected ion monitoring mode (GC-MS-SIM). Cell pellets were spun in a speedvac concentrator (12 mbar; Savant AES 1000) and weighed. Cholesterol, non-cholesterol sterols, and oxysterols were extracted using chloroform. After alkaline hydrolysis, the concentrations of cholesterol precursors were measured with GC-MS-SIM as previously described [5 (link),87 (link)]. The trimethylsilyethers of the (oxy)sterols were separated on a DB-XLB (30 m length × 0.25 mm internal diameter, 0.25 μm film) column (Agilent Technologies) using the 6890 N Network GC system (Agilent Technologies). Epicoprostanol (Steraloids, Newport, RI, USA) was used as an internal standard, to quantify the non-cholesterol sterols (Medical Isotopes, Pelham, NH, USA) on a 5973 Network MSD (Agilent Technologies). Total cholesterol was measured by GC-FID on an HP 6890 GC system (Hewlett Packard, Waldbronn, Germany), equipped with a DB-XLB (30 m length × 0.25 mm internal diameter, 0.25 μm film) column (Agilent Technologies) using 5α-cholestane (Steraloids) as internal standard [32 (link),88 (link)].
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