The average anhepatic time was 19.8 ± 1.7 minutes. The bile
duct was connected via ligation over the stent. BM cells were collected from the long bones of the extremities of wild-type (WT) or KO mice, and 2 × 107 unfractionated cells were injected intravenously into lethally irradiated (9.5 Gy) WT or KO mice via the tail vein. Animals were used as liver graft donors more than 2 months after BM transplantation. Additionally, the replacement of BMDCs selleck compound in the liver was confirmed in GFP radiation chimeras. Syngeneic LT was performed with KO, WT, or chimeric animals as donors and with WT B6 mice or B6.CD45.1 mice as recipients with 24 hours of cold storage. The recipient animals were euthanized 1, 3, 6, 12, or 24 hours after reperfusion so that we could obtain serum and liver graft samples. All procedures in this study were performed according to the guidelines of Guide for the Care and Use of Laboratory Animals (National Institutes of Health) and were approved by the institutional animal care and use committee of the University of Pittsburgh. Liver samples were fixed in 10% formalin, embedded in paraffin, sectioned (6 μm), and stained with hematoxylin and eosin. Grafts were also embedded in an optimal cutting temperature compound,
and 6-μm cryosections were stained with anti-CD3 and anti-CD11b monoclonal antibodies with nuclear Hoechst staining. Sections were visualized with 上海皓元 an Olympus BX51 epifluorescence microscope, and two-dimensional images were digitized with an Olympus/Optronics (Goleta, CA) charge-coupled device camera, c-Met inhibitor which was interfaced with MagnaFire image capture software. Messenger RNA (mRNA) expression was quantified by SYBR Green real-time RT-PCR
with an ABI-Prism 7000 sequence detection system (PE Applied Biosystems)20 and with primers for B7-H117 and other inflammatory and death-related molecules. The expression of each gene was normalized to the β-actin mRNA content and was calculated with respect to a normal liver. Hepatocytes and hepatic NPCs were isolated from the liver grafts by the collagenase digestion method22 with some modifications.21 Briefly, each liver was perfused in situ via the infrahepatic inferior vena cava (initially with 20 mL of Ca+Mg+-free HBSS containing 5 mM ethylene glycol tetraacetic acid and 10 mM HEPES and then with 100 mL of HBSS containing 0.025% collagenase B, 5 mM HEPES, 56 mg of calcium dichloride, and 0.005% trypsin inhibitor). NPCs and parenchymal cells were liberated from the removed liver grafts, and the initial cell suspension was filtered through a 70-μm nylon mesh. Hepatocytes and NPCs were separated by low-speed centrifugation (5 × 45g for 5 minutes) and washed by high-speed centrifugation (150g for 10 minutes).