LX2 cell line treated with or without BTC, and with or without fatty acids (Oleic acid and Omega3), and subsequent gene expression

LX2 cells treated with or without BTC and with or without oleic acid or DHA Overall design: LX2 cells were obtained from SL Friedman (Mount Sani Medical School) (46). LX2 cells are activated human hepatic stellate cells; they were maintained in DMEM with 10% FCS containing penicillin and streptomycin. Cells were plated onto 100 mm plastic petri dishes ~100,000 cells/plate and treated with fatty acids (at 50 µM) in endotoxin-free BSA (at 20 µM) during the growth phase. Fatty acids [oleic acid (18:1 ?9) and DHA (22:6, ?3); Nu-Chek Prep] were used at 50 µM for 2- 48-hour cycle treatments. After this pre-treatment, cells were trypsinized, washed in PBS, counted and plated at 3000 cells/well in a 96-well plate. Cells were fed DMEM with 1 % FCS without or with betacellulin (human recombinant betacellulin (BTC), R&D Systems) for 72 hrs; the concentration ranged from 1.25 to 25 ng/ml. At the completion of BTC treatment, media was removed, washed with PBS and DNA/well was quantified using the CyQuant cell proliferation assay (Thermofisher); DNA fluoresces was quantified [excitation at 485 nm & emission at 530 nm. This experiment was repeated 3 times.