Naik Shalin H, O'Keeffe Meredith, Proietto Anna, Shortman Hubertus Hochrein Ken, Wu Li
Division of Immunology, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
Methods Mol Biol. 2010;595:167-76. doi: 10.1007/978-1-60761-421-0_10.
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).
三十年来，在粒细胞-巨噬细胞集落刺激因子（GM-CSF）培养体系中由单核细胞和早期祖细胞生成树突状细胞（DC）一直是体外生成DC的金标准。然而，最新证据表明，这些培养体系所代表的是迁移性和炎性DC亚群，而非稳态下的DC亚群。相比之下，有人描述了一种不同的培养方法，即将小鼠骨髓与fms样酪氨酸激酶3配体（flt3配体）一起培养9天。在此，我们详细描述这种方法，以生成CD8(+)、CD8(-)和浆细胞样DC在表型、功能和发育上的等效细胞。这包括从中国仓鼠卵巢细胞中生长和纯化重组flt3配体、分离骨髓细胞以及对各亚群进行表型特征分析。与从脾脏分离DC（每只小鼠500万个）相比，这种简单方法能够生成大量DC（每只小鼠6000万至1亿个）。
