标准件与常用件课程.docVIP

? Acknowledgments The authors would like to thank Johns Hopkins University for the TC-1 cells. This work was supported by a National Health Research Institutes intramural grant (IV-103-PP-22) and grants from the National Science Council, which were awarded to Y.C. Song (NSC 99-2321-B-400-004-MY3) and S.J. Liu (NSC 103-2321-B-400-008). Author Contributions Y.C.S. and S.J.L. designed the studies. Y.C.S. performed the research and analyzed the data. Y.C.S. and S.J.L. wrote the manuscript. Additional Information C57BL/6 mice were immunized subcutaneously (s.c.) once with 1 μ g of peptide mixed with or without 10 μ g of CpG adjuvant. After one week, splenocytes were harvested, and the response of IFN-γ -secreting cells was determined by ELISPOT after 48 h of peptide stimulation. Briefly, 2 × 105 splenocytes were incubated with 1 μ g/ml irrelevant peptide or RAH peptide in an anti-IFN-γ -coated polyvinylidene fluoride (PVDF) plate for 48 h. After incubation, the cells were removed, and a biotinylated anti-IFN-γ Ab (eBioscience, San Diego, CA, USA) was added to each well. The plates were incubated at 37 °C for 2 h. Following the addition of the avidin-HRP reagent (eBioscience, CA, USA), the assay was developed using a 3-amine-9-ethyl carbazole (AEC; Sigma-Aldrich, MO, USA) staining solution. The reaction was stopped after 4–6 min by placing the plate under tap water. The spots were counted using an ELISPOT reader (Cellular Technology Ltd., Shaker Heights, OH, USA). For RAH-specific T cell staining, spleens were harvested seven days after the immunizations, and RAH-specific CD8+ T cells were detected by tetramer staining using a PE-labeled RAH tetramer (Beckman Coulter, CA, USA) and a FITC-labeled anti-CD8 monoclonal antibody (mAb) (eBioscience, CA, USA). The stained RAH-specific CD8+ T cells were analyzed by flow cytometry. Supplementary information accompanies this paper at /srep Competing financial interests: The authors declare no competing financial interests. How to