\Generation of Mature Murine Monocytes from Heterogeneous Bone Marrow and Description of Their Properties
\Generation of Mature Murine Monocytes from Heterogeneous Bone Marrow and Description of Their Properties
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DOI：
10.1369/0022155411416007
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发表时间：
2011-09-01
影响因子：
3.2
通讯作者：
Braun-Dullaeus, Ruediger C.
中科院分区：
文献类型：
--
作者：
Francke, Alexander;Herold, Joerg;Braun-Dullaeus, Ruediger C.
单核细胞参与了广泛的生理和病理过程，其中许多是在小鼠模型中进行研究的。目前分离小鼠单核细胞的方法很少，导致细胞产量和纯度不令人满意。在这里，我们描述了一种新的方法来有效地区分大量成熟的炎性单核细胞和异种骨髓细胞悬液。从Balb/c和C57BL/6小鼠的股骨和胫骨冲洗分离骨髓细胞悬液，并添加巨噬细胞集落刺激因子(M-CSF)，在超低贴壁表面培养，以抑制黏附介导的成熟。在指定的时间点采集细胞，对分化过程进行时间线分析，随后进行广泛的表型鉴定，以验证其单核细胞特性。为了确认下游的兼容性，我们测试了典型的单核细胞行为。我们的方案获得了每只供体小鼠24+/-6x10(6)个分化细胞，比之前描述的外周血液分离方法获得的产量高10倍。分化的细胞由约47%+/-12%的单核细胞组成，其余为成熟巨噬细胞。我们通过消耗黏附的巨噬细胞将单核细胞纯度提高到86%+/-6%。我们的发现表明，骨髓来源的单核细胞(BMDM)是一种有吸引力的研究工具，例如研究先天性和获得性免疫系统、动脉粥样硬化和感染期间的细胞迁移。此外，骨髓间充质干细胞移植可用于在小鼠疾病模型中测试新的体内治疗方法。(组织化学细胞化学杂志59：813-825,2011)
Monocytes are involved in a wide range of physiological and pathological processes, many of which are studied in mouse models. Current protocols to isolate murine monocytes are few and result in unsatisfactory cell yield and purity. Here, we describe a novel approach to efficiently differentiate large numbers of mature inflammatory monocytes from heterogeneous bone marrow cell suspensions. Bone marrow cell suspensions were isolated by flushing femurs and tibias from Balb/c and C57Bl/6 mice, supplemented with macrophage colony-stimulating factor (M-CSF), and were cultured on ultra-low attachment surfaces to inhibit adherence-mediated maturation. Cells were harvested at indicated time points, underwent time-line analysis of the differentiation processes, and were subsequently extensively phenotyped to verify their monocytotic properties. In order to confirm downstream compatibility, we tested for typical monocyte behavior. Our protocol yielded 24 +/- 6 x 10(6) differentiated cells per donor mouse, 10-fold higher than yields obtained using previously described peripheral blood isolation methods. Differentiated cells consisted of approximately 47% +/- 12% monocytes, the rest being mature macrophages. We increased monocyte purity to 86% +/- 6% by depleting adherent macrophages. Our findings indicate that bone marrow-derived monocytes (BMDMs) are an attractive tool to study, for example, the innate and adaptive immune system, atherosclerosis, and cellular migration during infection. Moreover, BMDM transplantation could be used to test novel, therapeutic in vivo approaches in mice disease models. (J Histochem Cytochem 59: 813-825, 2011)