Hepa 1–6 (CRL-1830), the murine HCC cell line, was obtained from American Type Culture Collection (ATCC, USA) and maintained in DMEM (Biowest) supplemented with 10% FBS (Biowest). C57BL/6 J mice (6 to 8 weeks of age) were purchased from the Chinese Academy of Science and housed at the Animal Maintenance Facility of the Shanghai Medical College, Fudan University. All animal experiments were performed in conformity with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The Institutional Care of Experimental Animals Committee of Fudan University approved all animal protocols (Permit Number: SYXK 2009–0082).
Hepa 1–6 tumor bearing mice were established as following: mice were injected subcutaneously at the left flank with 3 × 106 Hepa 1–6 cells in 200 μL RPMI 1640 (Biowest) or 200 μL RPMI 1640 alone as control. Three groups of tumor bearing mice and control mice were established (12 mice in each group). Two weeks after inoculation, the mice with visible tumor were sacrificed and spleens were collected for isolation of Tregs using mouse regulatory T cell isolation kit (Miltenyi). In brief, CD4+ T cells were enriched by negative selection and then went through positive selection for CD25+ T cells. The purity of Tregs was monitored via FACS.
Hepa 1–6 tumor bearing mice were established as following: mice were injected subcutaneously at the left flank with 3 × 106 Hepa 1–6 cells in 200 μL RPMI 1640 (Biowest) or 200 μL RPMI 1640 alone as control. Three groups of tumor bearing mice and control mice were established (12 mice in each group). Two weeks after inoculation, the mice with visible tumor were sacrificed and spleens were collected for isolation of Tregs using mouse regulatory T cell isolation kit (Miltenyi). In brief, CD4+ T cells were enriched by negative selection and then went through positive selection for CD25+ T cells. The purity of Tregs was monitored via FACS.