Silica gel g plates

The following tissues were used for lipid and fatty acid analysis: plasma, liver, and two brain areas (cortex and midbrain). Lipids were extracted immediately by Folch’s method [87 (link)], and lipid extracts were stored at −20 °C under nitrogen atmosphere prior to further analysis. Non-polar lipids were separated using one-dimensional TLC on 10 × 10 cm² silica gel G plates (Merck KGaA, Darmstadt, Germany) in the solvent system hexane/diethyl ether/acetic acid (80:20:1, v/v/v). Polar lipids were separated by two-dimensional TLC on 10 × 10 cm² 1.2% boric acid-impregnated silica gel plates using chloroform/methanol/ammonium hydroxide (65:25:4, v/v/v) in the first dimension and n-butanol/acetic acid/water (90:20:20, v/v/v) in the second direction. Lipids were identified by reference to authentic standards and by using specific colour reagent [88 ]. In addition, several lipid identifications were performed using HPLC-MS-MS (details can be found in our previous publication [48 (link)]. Plates were sprayed with 0.2% (w/l) 8-anilino-4-naphtholenesulphonic acid in dry methanol and viewed under U.V. light to reveal lipids. Individual lipids were scraped from the plates and their fatty acid compositions and contents were determined by gas chromatography (GC) with heptadecanoate (C17:0) as an internal standard.

DCs (6 × 10⁶/mL, in RPMI 1640, 10% FCS) were labelled in Petriperm dishes with 0.125 μCi/mL [¹⁴C] AA (Amersham, Buckingham, UK) overnight. At the end of the incubation, cells were washed twice and resuspended in RPMI 1640 supplemented with 0.2% fatty acid free bovine serum albumin (Sigma). DCs were stimulated for 3 h and the reaction was terminated by the addition of 2 mL of chloroform/methanol/formic acid (1 : 2 : 0.2, v/v/v, all from Sigma-Aldrich) followed by agitation. Then, 1 mL of water and 2 mL chloroform were added. Chromatographic separation of lipids was performed by evaporating the organic phase under a stream of nitrogen, redissolving the residue in chloroform, and loading the extract on silica gel G plates (Merck, Darmstadt, Germany). Fatty acids were separated by thin layer chromatography using hexane/ethyl ether/formic acid (15 : 10 : 1, v/v/v, all from Sigma-Aldrich) as a solvent system for 30 min. AA position on TLC plates was determined as comigration with commercially available standard after exposure to iodine vapors. Autoradiography of TLC plates was performed using a phosphoimaging system (FLA 2000, Fuji). The results are expressed as the percentage of radioactivity in the arachidonic acid band on the total radioactivity recovered from each lane.