Collagen coated 12 well plates

Mouse primary hepatocytes were isolated as described previously [9 (link),36 (link),37 (link)]. Hepatocytes were seeded in collagen-coated 12-well plates (Becton, Dickinson and Company, Franklin Lakes, NJ) at a density of 4 × 10⁵ cells/well and cultured in William’s E medium (Thermo Fisher Scientific) with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin, Gemini Bio Products, West Sacramento, CA). Sixteen hours after seeding, the cells were treated with medium containing 100 μM CDCA, 100 μM Wy, CDCA/Wy (each 100 μM) or dimethyl sulfoxide (DMSO) as a vehicle, respectively. For RXRα inhibition using HX531, the cells were treated with 1 μM HX531 (Tocris Bioscience, Bristol, UK) 4 h after seeding. Twelve hours after HX531 treatment, the cells were treated with medium containing 100 μM CDCA, 100 μM Wy, CDCA/Wy (each 100 μM) or dimethyl sulfoxide (DMSO) with or without 1 μM HX531, respectively. Three days after treating the cells with the compounds, they were harvested and subjected to measurement mRNA levels.

Primary hepatocytes were isolated from C57BL/6J mice as previously reported (Seglen, 1976 (link)) and seeded at a density of 2 × 10⁵ cells on collagen-coated 12-well plates (Becton Dickinson and Company, Franklin Lakes, NJ) in Williams’ Medium E (Thermo-Fisher Scientific, Waltham, MA) supplemented with 5% FBS and penicillin/streptomycin/amphotericin B solution (Gemini Bioproducts, West Sacramento, CA).

previously reported [8 (link)] and seeded on collagen-coated 12-well plates (Becton Dickinson and Company) at a density of 2 × 10⁵ cells in Williams’ Medium E (Thermo-Fisher Scientific, Waltham, MA) supplemented with 5% FBS and penicillin/streptomycin/amphotericin B solution (Gemini Bio-products). The cells were harvested and subjected to qRT-PCR for mRNA quantification.