Pan dc isolation kit

T cells, B cells, NK cells, and DCs were isolated from the spleen using a Pan T Cell Isolation Kit II, a Pan B Cell Isolation Kit II, a Macrophage Isolation Kit, and a Pan DC Isolation Kit, respectively (Miltenyi Biotec, Bergisch Gladbach, Germany). Neutrophils were isolated from bone marrow cells using the Percoll gradient (Sigma-Aldrich, St. Louis, MO, USA), as described elsewhere (37 (link), 38 (link)). Briefly, bone marrow cells were extracted from mouse femurs and tibias. After removal of red blood cells by the ACK buffer (Beyotime, Shanghai, China), cells were carefully loaded on a 52, 69, 78% percoll (GE Healthcare) gradient, and centrifuged at 1500 g for 30 min without breaking. Cells were isolated on the 69/78% interface layer. In some experiments, peritoneal neutrophils were isolated after CLP surgery. Pasteur pipettes were filled with cold PBS and inserted through the peritoneal wall to inject PBS into each mouse. After aspiration of the fluid from the peritoneum, the peritoneal fluid was slowly withdrawn. The cell suspension was then centrifuged at 1700 rpm for 5 min and washed with PBS.

Flt3L-expressing B16 cells were subcutaneously injected into C57BL/6 mice (3.5 × 10⁵ cells/mouse) in order to expand the DC population^{40 (link)}. After approximately 2 weeks, the tumor-bearing mice were sacrificed and the spleens were harvested for DCs. Spleens were digested with 1 mg/mL collagenase and 10 µg/mL DNase I (Roche), and red blood cells lysed with ACK lysis buffer. Splenic DCs were purified using the Pan DC Isolation Kit (Miltenyi), according to the manufacturer’s protocol.