Cd4 or cd8 t cell isolation kit

For preparation of naïve T cell subsets, CD4⁺CD62L⁺ or CD8⁺CD62L⁺ naïve T cells were isolated from lymph nodes using a CD4⁺ or CD8⁺ T cell isolation kit (Miltenyl Biotec, USA). Preparation of T cell blasts and Th1, Th17 and Tc1 cells was performed as described previously (Fang et al., 2010 (link)). Briefly, CD4⁺ T cells or CD8⁺ T cells were enriched by using CD4⁺ or CD8⁺ T cell isolation kit (Miltenyl Biotec) from lymph nodes of 6- to 8-week age LIME^+/+ or LIME^-/- mice according to the manufacturers’ instructions. As determined by flow cytometry, > 95% of the enriched cells were CD4⁺ or CD8⁺ cell. Enriched CD4⁺ T cells were cultured at a concentration of 1 × 10⁶ cells/ml in complete RPMI medium supplemented with various cytokines and anti-cytokine mAb as described in Supplementary Materials and Methods. The non-polarized T cell blast (B) and the differentiated effector T cells are harvested after 24 h and 5 days, respectively.

The 10⁸ GL261 cells were collected and resuspended in DMEM. These cells were then frozen in a −80°C freezer for 1 h and thawed at 37°C for three cycles to lyse the cells. The solution was centrifuged at 300 rpm for 30 min, and the supernatants were collected and stored at 4°C for further use as tumor-specific antigens.
The mice were euthanized, and the spleens were collected. The spleens were disassociated by squeezing, and all released and suspended cells were collected. The suspended cells were treated with red cell lysis buffer (Beyotime), and the splenocytes were centrifuged at 400 g for 3 min. The splenocytes were cultured with GL261 cell lysates (1:3 for the cell numbers) for 48 h. The lymphocytes were isolated from cultured splenocytes using gradient centrifugation on histopaque-1077. CD4⁺ T and CD8⁺ T cells were isolated from lymphocytes according to the instructions of CD4⁺ or CD8⁺ T cell isolation kits (Miltenyi Biotec).
GL261 cells expressing a control shRNA or H2-K^D shRNA (MHC-I subunit) were infected with lentivirus expressing PTEN shRNA. CD4⁺ or CD8⁺ T cells were co-cultured with GL261 cells at a ratio of 3:1 for 24 h, and CD4⁺ or CD8⁺ T cells were then collected and subjected to ELISpot analysis with an ELISpot kit (R&D Systems) containing plates coated with an anti–IFNγ or an anti–IL-2 antibody.