Petroleum ether diethyl ether acetic acid

Huh-7 cells were incubated in DMEM supplemented with 10% foetal calf serum and PSG containing 0.4 µCi per well of [¹⁴C]oleic acid for 24 hours, followed by lipid extraction and separation by TLC in hexane/diethyl ether/acetic acid (70/30/1, v/v). Radioactivity of TG fraction was measured.
HepG2 cells were incubated with MEM w/o FBS +6 µCi/mL [³H]glycerol (PerkinElmer) +1.5 mM glycerol (Sigma-Aldrich)+1% BSA (Sigma-Aldrich) for 5 hour. Cells were collected and washed with PBS to remove the excess of medium. Lipids were extracted adding 3 mL of chloroform:methanol (2:1 v/v) (Sigma-Aldrich) and 1 mL of acidified solution (17 mM NaCl, 1 mM H₂SO₄) (Sigma-Aldrich). Samples were centrifuged at 3000 rpm for 10 min. The lower organic phase was saved and dried under a gentle nitrogen stream. Lipids were reconstituted in 50 µL of chloroform (Sigma-Aldrich), and separated by one-dimensional TLC using TLC silica gel plates (Merck-Millipore). Triolein (Sigma-Aldrich) was used as a marker. Petroleum ether:diethyl ether:acetic acid (40:60:1, vol/vol) (Sigma-Aldrich) was used as the mobile phase. The area of TLC silica gel corresponding to the newly synthesised radio-labelled TG were cut out. The rate of released radio-labelled TG was measured, using liquid scintillation counting (PerkinElmer). Data were normalised for number of cells.

Cer and C1P measurements were achieved as described in Bini et al. [39 (link)]. Briefly, cells were labeled with 40 μCi/ml of [³H] palmitate (Perkin Elmer, Connecticut, USA) for 24h in serum–free medium in the presence or absence of agonists. Cells were then washed with ice-cold calcium-free phosphate-buffered saline (PBS) and scraped into methanol and chloroform (2:1) (Sigma Aldrich, Milan, Italy). Lipids were extracted by separation of phases as described in [39 (link)]. chloroform phases were dried, and lipids were separated by thin-layer chromatography (TLC) using silica gel 60-coated glass plates (Merck, Darmstadt, Germany). The plates were developed for 50% of their lengths with chloroform/methanol/acetic acid (9:1:1, v/v/v) and then dried. They were then developed for their full length with petroleum ether/diethylether/acetic acid (60:40:1, v/v/v) (Sigma Aldrich, Milan, Italy). The position of Cers was identified after staining with I₂ vapor by comparison with authentic standards. Radioactivity of the samples, obtained by scraping the Cer and C1P spots from the plates, was quantified by liquid scintillation counting. CerK activity was assayed as described previously [39 (link), 47 (link)] with some modifications.