Dnase

Total RNA was extracted from isolated hepatocyte as described above. To remove genomic DNA contaminants, 5μg total RNA was treated with 2.5 units DNAse (Roche Applied Science, Mannheim, German) for 15 minutes at 20°C followed by inactivation of DNAse enzyme at 70°C for 8 minutes. Then, cDNA was synthesized from total RNA using SuperScript® III First-Strand Synthesis System (Invitrogen) according to the manufacturer’s protocol. 20ng cDNA was loaded into each well in RT2 Profiler 96-well PCR array plates (PARN-006, QIAGEN, Valencia CA) and amplified using the ABI 7500 real-time PCR System. The PCR reaction was programmed as follows: initial denaturing at 95°C for 10 min followed by 95°C for 15 sec, 60°C for 1 min, cycled 40 times. The median cycle threshold value (Ct) was uploaded onto the SABioscience website (http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php) and the fold change of each gene expression was calculated using the provided software according to manufacturer’s instruction. Additionally, subsets of genes with the values of fold change larger than 1.5 across the 3 comparisons (normal vs compensated cirrhotic hepatocytes, normal vs decompensated cirrhotic hepatocytes, compensated vs decompensated cirrhotic hepatocytes) were used to find possible signal pathways using Ingenuity Pathway Analysis software.

Lymphocytes were isolated from mesenteric lymph nodes, lung, small intestine and bone marrow. For lamina propria (LP) lymphocyte isolation, tissue was digested for 90 minutes at 37°C in 0.75ml complete media (CM) containing 100mg/ml collagenase D (Roche), 1mg/ml dispase II (Invitrogen), and 0.2 mg/ml DNAse (Roche). LP lymphocytes were further washed with CM and enriched by 33% Percoll gradient centrifugation. For lung lymphocyte isolation, the lung was perfused by injecting 10ml PBS into the right ventricle of the heart. Further, lung tissue was incubated at 37°C for one hour in CM containing 1mg/ml collagenase D (Roche) and 0.15mg/ml DNAse (Roche). Cells were washed, filtered through 40µm cell strainer and treated with red blood cell lysis buffer. To collect BAL fluid the mouse lungs were lavaged three times with 1 ml of PBS, and cells were centrifuged at 350g for 5 minutes and stained for flow cytometry.

Tumours were mechanically dissociated and digested in 1mg/ml collagenase D (Roche), 1mg/ml collagenase A (Roche) and 0.4mg/ml DNase (Roche) in PBS, at 37°C for 2hs. Lymph nodes were mechanically dissociated and digested with 1mg/ml collagenase A (Roche) and 0.4mg/ml DNase (Roche) in PBS, at 37°C. After 30 mins, collagenase D (Roche) was added (final concentration of 1mg/ml) to lymph node samples and digestion was continued for a further 30 mins. EDTA was added to all samples to neutralise collagenase Activity (final concentration (5mM) and digested tissues were passed through 70μm filters (Flacon) ready for staining. 5ml of Red Blood Cell Lysis (RBC) lysis buffer (150mM NH₄Cl, 1mM KHCO₃, 0.1mM EDTA) was added to blood samples for 5 mins and neutralized with 45ml of PBS.