Data acquired by FACSCalibur (BD Biosciences) were analyzed using FlowJo software (Tree Star, Ashland, OR). Mice were intraperitoneally injected

with 5 mg of αGalCer (Alexis Biochemicals, Lausen, Switzerland) diluted in PBS/0.05% Tween (BDH, Radnor, PA) or vehicle only. Two hours postinjection, organs were harvested for flow cytometric analysis and serum was collected for IL-4 determination by enzyme-linked immunosorbent assay (ELISA) using the Mouse IL-4 ELISA Ready-Set-Go kit (eBioscience) following the manufacturer’s instructions. RPMI this website 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, Logan, UT), 100 U/mL penicillin, 100 mg/mL streptomycin (Invitrogen), and 50 mM 2-mercaptoethanol (Sigma) was used for T-cell culture. Splenocytes were labeled with 5 mM 5-(and-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE; Invitrogen) and stimulated with 5 mg/mL plate-bound anti-CD3

(clone 145-2C11) and 5 mg/mL anti-CD28 (clone 37.51) in suspension (WEHI, Melbourne, Australia). After 48 hours proliferation was assessed by flow cytometry. learn more Viable CFSE-labeled T cells were gated using 7-AAD and CD4-APC or CD8-APC (BD Biosciences) staining and the percentage of dividing cells was determined using the FlowJo proliferation platform. Liver donor mice were intraperitoneally injected for 3 consecutive days with 0.6 mg of monoclonal anti-CD4 (clone GK1.5) antibody. Donor livers were transplanted at day 6 and spleens were harvested to confirm depletion by flow cytometry using CD4-PerCP-Cy5.5 antibody (clone L3T4) (BD Biosciences). Four-week-old recipient mice were irradiated twice for 20 minutes with 550 rads, 2 hours apart. The donor bone marrow was extracted from tibia and femur and resuspended in sterile saline; 106 cells were injected into the tail vein of irradiated recipients. After 6 weeks of reconstitution, chimeric mice were sacrificed and organs and blood were harvested for flow cytometric analysis. Donor livers were used for

orthotopic transplantation into WT recipient mice. Donor livers were subjected to prolonged cold storage in University of Wisconsin (UW) solution as described.25 find more Male donors and recipients were used. Donor livers were dissected, the bile duct was cannulated, and the gall bladder was removed. The liver was perfused through the portal vein with UW solution and was excised. The liver graft remained in UW solution for 18 hours at 4°C. Livers of recipient mice were removed and the graft liver was placed in the orthotopic position. The cuff technique was used for anastomosis of both the portal vein and the infrahepatic vena cava. The suprahepatic vein was anastomosed. The bile duct was reconnected through the stent. The anhepatic time was kept below 25 minutes.