Following perfusion, the liver was withdrawn and hepatocytes were collected by disrupting the liver capsule and filtering the resulting cell suspension through a 100 μm cell strainer (BD biosciences). Cells were washed twice by centrifugation and resuspension in washing buffer. Cell yield and vitality were determined by trypan blue staining (Fluka) using a Neubauer counting chamber (Brand). Preparations with vitality greater than 70% were employed for experiments. For each experiment cells from a single animal, or, if necessary, from a cell pool from up to three animals were used. For all experiments primary mouse hepatocytes were cultivated at sub-confluence on collagen I- coated cell ware. Two million cells per dish (diameter: 10 cm) were seeded in 5 ml of full medium (phenol red-free Williams E medium (Biochrom) supplemented with 10% (v/v) FBS (Invitrogen), 100 nM dexamethasone, 10 μg/ml insulin, 2 mM L-glutamine, and 1% (v/v) penicillin/streptomycin 100x (both Invitrogen)). Following plating, cells were allowed to attach for 4 h. Subsequently, hepatocytes were washed, twice vigorously and once gently, with PBS (PAN Biotech) to remove unattached cells, and cultured in serum-free cultivation medium (phenol red-free Williams E medium supplemented with 100 nM dexamethasone, 2 mM L-glutamine, and 1% (v/v) penicillin/streptomycin 100 x) for 24 h prior to experiments. Before stimulation an additional 4 hour time interval in starvation medium(phenol red-free Williams E medium supplemented with 2 mM L-glutamine and 1% (v/v) penicillin/streptomycin 100 x) was applied
Growth protocol
Eight to twelve weeks Male C57BL/6N mice were employed for primary hepatocyte isolation
Extracted molecule
total RNA
Extraction protocol
RNA was extracted at 1,2,3,4,5,6,7,8,9,10,12,24 hours under stimulated conditions and additionally at -5, 0, 4, 8, 12, 24 hours for controls using the RNeasy Mini Plus Kit (Qiagen, Hilden, Germany).
Label
Biotin
Label protocol
Biotinylated cRNA was generated from 1.5 µg total RNA using the One-Cycle Target Labeling Assay (Affymetrix).
Hybridization protocol
15 µg labelled cRNA were hybridized onto the Mouse Genome 2.0 GeneChip® Arrays (Affymetrix, CA, USA) for 16h at 45°C using Hybridization Wash and Stain Kit (Affymetrix)
Scan protocol
Arrays were washed and stained with GeneChip Fluidics Station 450 and scanned with GeneChip Scanner 3000 using the GeneChip Operating Software (Affymetrix).
