YFP positive cells were obtained by liver perfusion method from mice treated with DDC diet (n=4) for 5 weeks. Livers were perfused with EGTA solution (NaCl 137 mM, KCl 5.4 mM, NaH2PO4H2O 0.64 mM, Na2HPO4 0.85 mM, HEPES 1mM, NaHCO3 4.17 mM, EGTA 0.5 mM and glucose 5mM, pH= 7.4) at a flow rate of 5ml/min until liver gets pale. After that, the liver was perfused with collagenase solution (NaCl 137 mM, KCl 5.4 mM, NaH2PO4H2O 0.64mM, Na2HPO4 0.85 mM , HEPES 1 mM, NaHCO3 4.17 mM and CaCl2·2H2O 3.8 mM, pH=7.4) containing Collagenase A 0.5 g/L (Roche Diagnostics GmbH, Manheim, Germany) at a flow rate of 5 mL/min for 11 minutes. The digested liver was minced in Petri dish with ice cold 1x Hank’s Balanced Salt Solution (HBSS) (Biological Industries) and filtered through 70-μm cell strainer (BD Biosciences, Erembodegen, Belgium). The undigested liver was re-digested with collagenase solution containing 0.5 g/L Collagenase A, 0.5 g/L Pronase and 50 mg/L DNAse I (Roche Diagnostics GmbH) and stirred for 30 minutes at 37°C before liver homogenates were filtered using a 70-μm cell strainer. After slow centrifugations at 100g for 2 minutes the supernatant was centrifuged at 300g for 5 minutes and the pellet containing non-parenchymal cells were sorted on a FACSAria (BD Biosciences). YFP+ gate was established based on wild type mice. Dead cells were excluded by Live/Dead cell stain kit (Life Technologies, Eugene, Oregon) and YFP+ cells were collected in RLT buffer.
Extracted molecule
total RNA
Extraction protocol
RNA from sorted cells was obtained using the RNeasy Micro Kit (Qiagen GmbH, Hilden, Germany). RNA from whole liver samples was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. Total RNA was retrotranscribed using a high-capacity complementary DNA reverse-transcription kit (Applied Biosystems, Foster City, CA) and amplified using Platinum® SYBR® Green qPCR SuperMix-UDG (Life Technologies) in a 7900 HT instrument (Applied Biosystems).
Label
biotin
Label protocol
Standard Affymetrix protocol
Hybridization protocol
Standard Affymetrix protocol
Scan protocol
Standard Affymetrix protocol using a Gene chip scanner 3000
Description
YFP positive cells obtained from mice treated with DDC (replicate 2)
Data processing
Data processing cel files were normalized with 'rma' function of the Bioconductor package (v2.12) ' R.3.0.0
