The same quantity MK-8669 chemical structure of total RNA was reverse-transcribed to complementary DNA (cDNA)

using M-MLV Transcriptase (Invitrogen) in the presence of oligo-dT primers (Shenggong, China). Quantitative PCR was performed using SYBR Green I (Takara) for 45 cycles at 15 seconds at 95° and 60 seconds at 60° with Rotor-Gene 6000 (Corbett Research) according to the manufacturer’s instructions. Quantitative PCR primers were included in Supporting Information materials. Results were analyzed by ΔΔCt method as described before.28 Values were expressed as fold change in comparison with control. Donor splenocytes were isolated from biweekly CCl4-treated C57BL/6 and HBV-tg mice. Four million splenocytes were adoptive transferred weekly (i.p.) to Rag1−/− recipient mice from PD98059 cell line the same genetic background and age for 4 weeks. The recipient Rag1−/− mice were

sacrificed and liver tissues were collected 72 hours after the fourth transfer. HSCs were isolated from liver of HBV-tg mice by collagenase and pronase perfusion and 8.2% Nycodenz (Sigma) gradient centrifugation.29 For isolation of hepatic NKT cells, first, HBV-tg mice were injected CCl4 (i.p. 0.5 μL of body weight) 12 hours and isolation of liver mononuclear cells (MNCs). Liver MNCs were stained for PE-NK1.1 and APC-CD3 (BD PharMingen, San Diego, CA) and sorted by flow cytometry (Becton Dickinson) for CD3+NK1.1+ NKT cells. For the coculture experiment, HSCs were previously cultured for 24 hours before constitution of NKT cells, and then cocultured (1:10) with CCl4-pretreated NKT cells for another 24 hours with or without functional purified neutralizing cytokine antibodies of IL-4, IL-13, or IFN-γ at a concentration of 5 μg/mL (eBioscience).

After coculture, NKT cells were removed by washing, HSCs were visualized with phase-contrast microscopy, and collected by mild trypsinization check details for analyzing the transcription of α-SMA. HSCs RNA extraction were using RNAprep pure Micro Kit (Tiangen Biotech, Beijing, China). Analysis of liver transaminase activity, liver histology, and immunohistochemistry for α-SMA, liver mononuclear cell (MNC) isolation and flow cytometric analysis, cell depletion, NKT cell preparation, and adoptive transfer to Rag1−/− mice in vivo CD1d block methods are included in the Supporting Information Materials online. Student t test was chosen to compare values between two groups. Analysis of variance (ANOVA) was used to compare values from multiple groups. Data are expressed as means ± standard deviation (SD). P < 0.05 was considered statistically significant. It was found that HBV-tg mice had an elevated level of serum ALT at the age of 2, 3, 4, and 6 months than that of normal C57BL/6 mice, showing that ALT levels were much higher in HBV-tg mice compared with C57BL/6 mice (all below 40 IU/L) (Fig.