CHAPTER 7 – Isolation, culture and propagation of dendritic cells

Monocyte-derived dendritic cells (MDDCs) are the most commonly used type of human DC, but are only rarely prepared from mice largely because the yield is small, and there are other easier methods available to produce mouse DCs. Blood is obtained by cardiac puncture. PBMCs are separated by density gradient centrifugation and plated at 8–10 million cells/well in a six-well plate. The mouse peritoneal cavity harbors a small resident population of myeloid cells that are generally regarded as being macrophages. Peritoneal contents are recovered by gentle lavage. Mature mouse DCs will differentiate from fetal liver progenitor cells using a stromal cell feeder layer, GM-CSF, Flt-3L, and SCF to induce differentiation, followed by GM-CSF plus TNFα to induce maturation. DCs isolated from mouse myeloma or GM-CSF and CD40L-transduced colon carcinoma cells will activate tumor-specific T cells without exogenous antigen loading.