Engineered human hepatocyte organoids enable CRISPR-based target discovery and drug screening for steatosis
penicillin/streptomycin solution. To this medium we added all components present in HEP medium, with the exception of 15% RSPO1-CM , to avoid the presence of unlabeled glucose from this medium in the culture medium. Organoids were plated into fresh BME droplets and cultured in this medium for 1, 3 or 5 days. Organoids were harvested in cold SILAC medium, washed twice with the same medium and dry organoid pellets were snap-frozen in liquid nitrogen.
. Lipid extracts were kept frozen under a nitrogen atmosphere until analysis. Neutral lipid analysis was performed on an ACQUITY Premier BEH C18 column . Elution was performed at 60 °C using a binary gradient from methanol:water to methanol:isopropanol:ethyl acetate . Both solvents contained 25 mM ammonium formate. Gradient composition was : ; ; ; ; and the flow rate was kept constant at 0.4 ml min.
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