Anti mouse dx5 microbeads

Splenocytes were isolated from C57BL/6 or BALB/c mice. The granulocytes and RBCs were removed by Ficoll (Sigma-Aldrich) density gradient centrifugation. After the depletion of CD3ɛ⁺ and DX5⁺ cells from the cell population using anti-mouse CD3ɛ and anti-mouse DX5 microbeads, the CD11b⁺ cells and B220⁺ cells were purified using anti-mouse CD11b and anti-mouse B220 microbeads (all from Miltenyi Biotec). Isolated B cells and monocytes (1:1 ratio mixture) were transduced with the indicated adenoviral vector in a 9-min, 2,800-r.p.m. centrifugation step at room temperature, and the cells were subsequently incubated for an additional 18 h (for the B/Mo/αGC preparation this step was skipped). αGC (KRN7000, Enzo Life Science, Japan) was loaded into the prepared B cells and monocytes for NKT activation based on our previous studies20 (link)21 (link)22 (link)51 (link).

Different lymphocyte subsets were purified from splenic mononuclear cells isolated from Balb/c mice (Charles River Laboratories, Wilmington, MA, USA). If necessary, further isolation was performed by magnetic activated cell sorting (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany) or by FACS (FACSAria I, BD Biosciences, San Jose, USA).
Briefly, cell suspension of the spleen was prepared by cutting small pieces and gently pressing through a 100 μm wire mesh. DX5⁺ cells were purified using anti-mouse-DX5⁺ MicroBeads (Miltenyi Biotec). Cells were passed through a MACS column (type LS) attached to a MidiMACS magnet (Miltenyi Biotec). DX5⁺ cells were collected in the positive fraction. DX5⁺ splenocytes were labeled with FITC-conjugated anti-mouse CD3 molecular complex (clone: 17A2, rat IgG2b) and PE-conjugated anti-mouse CD49b (clone: DX5, rat IgM) (all from BD Biosciences) for further DX5⁺NKT cell isolation by FACS sorting. CD4⁺CD62L^high and CD4⁺CD62L_low cells were purified using the CD4⁺CD62L⁺ Isolation Kit (Miltenyi Biotec) and CD8⁺ cells by using anti-mouse-CD8⁺ MicroBeads (Miltenyi Biotec).