CD8+, CD8-, and plasmacytoid dendritic cell generation in vitro using flt3 ligand

The generation of dendritic cells (DCs) from monocytes and early progenitors in GM-CSF cultures has been the gold standard for in vitro generation of DCs for three decades. However, the most recent evidence suggests that these cultures represent the migratory and inflammatory DC subtypes and not the DC subtypes found in the steady state. By contrast a different culture method was described where mouse bone marrow is cultured with flt3 ligand for 9 days. Here, we describe this method in detail for the generation of the phenotypic, functional, and developmental equivalents of CD8(+), CD8(-), and plasmacytoid DCs. This includes growth and purification of recombinant flt3 ligand from Chinese hamster ovary cells, isolation of bone marrow cells, and phenotypic characterization of the subsets. This simple method allows generation of large numbers of DCs (60-100 million from one mouse) compared to splenic DC isolation (5 million per mouse).