Bone Marrow-Derived Macrophages (BMM): Isolation and Applications.
Bone Marrow-Derived Macrophages (BMM): Isolation and Applications.
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DOI：
10.1101/pdb.prot5080
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发表时间：
2008-12-01
期刊：
影响因子：
--
通讯作者：
Porse, Bo
中科院分区：
文献类型：
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作者：
Weischenfeldt, Joachim;Porse, Bo
引言
骨髓来源的巨噬细胞（BMM）是原代巨噬细胞，在生长因子存在的情况下由骨髓细胞体外衍生而来。巨噬细胞集落刺激因子（M - CSF）是一种谱系特异性生长因子，负责定向髓系祖细胞增殖和分化为巨噬细胞/单核细胞谱系的细胞。缺乏功能性M - CSF的小鼠巨噬细胞和破骨细胞缺乏，并患有骨硬化症。在本实验方案中，骨髓细胞在含有M - CSF的培养皿中生长，M - CSF由L929细胞分泌，并以L929条件培养基的形式使用。在这些条件下，骨髓单核细胞/巨噬细胞祖细胞将增殖并分化为同质的成熟BMM群体。通过对Mac - 1和F4/80表面抗原表达进行荧光激活细胞分选（FACS）分析来评估分化效率。一旦分化，BMM适合多种类型的实验操作，包括形态学、基因表达和生理学研究。例如，巨噬细胞等吞噬细胞具有摄取微生物的独特能力。我们描述了一种通过将BMM暴露于荧光标记的酵母聚糖生物颗粒来检测其吞噬效率的测试方法。此外，还描述了一种将DNA或小干扰RNA（siRNA）递送至这些难以转染细胞的方法。最后，使用羧基荧光素琥珀酰亚胺酯（CFSE）测定BMM的增殖，CFSE是一种荧光素衍生物，在细胞分裂后在子细胞之间平均分配。
INTRODUCTIONBone marrow-derived macrophages (BMM) are primary macrophage cells, derived from bone marrow cells in vitro in the presence of growth factors. Macrophage colony-stimulating factor (M-CSF) is a lineage-specific growth factor that is responsible for the proliferation and differentiation of committed myeloid progenitors into cells of the macrophage/monocyte lineage. Mice lacking functional M-CSF are deficient in macrophages and osteoclasts and suffer from osteopetrosis. In this protocol, bone marrow cells are grown in culture dishes in the presence of M-CSF, which is secreted by L929 cells and is used in the form of L929-conditioned medium. Under these conditions, the bone marrow monocyte/macrophage progenitors will proliferate and differentiate into a homogenous population of mature BMMs. The efficiency of the differentiation is assessed using fluorescence-activated cell sorting (FACS) analysis of Mac-1 and 4/80 surface antigen expression. Once differentiated, the BMMs are suitable for numerous types of experimental manipulations, including morphological, gene expression, and physiological studies. For example, phagocytic cells such as macrophages have a unique ability to ingest microbes. We describe a test for the phagocytic efficiency of BMMs by exposing them to fluorescently labeled yeast zymosan bioparticles. Also, a method to deliver DNA or small interfering RNAs (siRNAs) into these hard-to-transfect cells is described. Finally, the proliferation of the BMMs is assayed using carboxyfluorescein succinimidyl ester (CFSE), a fluorescein derivative that partitions equally between daughter cells after cell division.