Hcm singlequot

Primary human hepatocytes were obtained from Lonza and cultured in Hepatocyte Culture Media (HCM), consisting of Hepatocyte Basal Media (Lonza) supplemented with Lonza’s HCM SingleQuot, as described previously (26 ). Briefly, cells were plated at a density of 1 × 10⁵ cells/ml and allowed to attach overnight prior to treatment with HDL NPs. Cells were treated with 20 nM or 50 nM HDL NPs, 50 nM human HDL or PBS for 72 or 120 h. Lysates were prepared using the mammalian protein extraction reagent (MPER) cell lysis buffer, and Western blotting analyses for GPX4 and β-actin conducted as described above. For the C11-BODIPY assay, the primary human hepatocytes were treated for 72 h prior to addition of the C11-BODIPY reagent and subsequent analysis by flow cytometry.

Differentiation of hepatocyte-like cells was performed as described previously (Si-Tayeb et al., 2010 (link)). Briefly, hESCs were plated on matrigel-coated plates as single cells and cultured in the presence of ROCK inhibitor for 24 hr. After reaching 80% confluency, differentiation was initiated by adding RPMI medium supplemented with B27 (GIBCO, Life Technologies), 0.5% nonessential amino acids (Life Technologies) and Activin A (100 ng/ml, R&D Systems) for 5 days to induce definitive endoderm stage cells. Cells were further differentiated by exposure to RPMI medium supplemented with B27, 0.5% nonessential amino acids, FGF4 (10 ng/ml, R&D Systems) and BMP4 (20 ng/ml, R&D Systems) for 5 days. After an additional 5 days of cultivation in RPMI/B27 supplemented with 0.5% nonessential amino acids and HGF (20 ng/ml, Peprotech), cultures mainly consisted of immature hepatocytes. Maturation to hepatocyte-like cells was initiated by cultivation in HBM medium (Lonza, UK) supplemented with HCM SingleQuots (Lonza) and Oncostatin M (20 ng/ml, R&D Systems). Hepatocytes were probed for cell-specific markers, such as α-fetoprotein (AFP) and hepatocyte nuclear factor 4α (HNF4α).