Silica gel 60 plastic sheets

Albumin-oleate complex was prepared according to [23 (link)] with slight modifications. In brief, radiolabeled oleic acid [9,10-³H]; 50 Ci/mmol; (Hartmann Analytic, Braunschweig, Germany) was dried in a glass tube under a stream of nitrogen. 10 μL of non-radiolabeled 20 mmol/L sodium oleate (Merck, Vienna, Austria) was added per 1 μCi dried [³H] oleic acid followed by vortexing. Finally, an equal volume of 20% fatty acid-free BSA solution (Merck, Vienna, Austria) was added and vortexed. EA.hy 926 cells were incubated with 100 μg/mL EV-HDL or EL-HDL for 5 or 16 h, washed with PBS and pulsed with [³H] oleic acid/ BSA for 30 min as described [24 (link)]. Cells were washed twice with ice-cold PBS and extracted with hexane/isopropanol (3:2, v/v), extracts evaporated in the SpeedVac and redissolved in chloroform before TLC using Silica Gel 60 plastic sheets (20×20 cm; Merck, Austria) and a solvent system for the separation of neutral lipids (hexane/diethyl ether/acetic acid, 70:29:1, vol/vol). After primuline staining lipids were visualized using an UV lamp (366 nm) and [³H]CE and [³H]tria-cylglycerol (TAG) spots were cut out for scintillation counting. Following lipid extraction, cells were lysed with 0.3 mol/L NaOH/0.1% SDS, and the protein concentrations were determined according to [25 (link)].