Methods used to prepare bone marrow-derived macrophages (BMDMs) may influence the outcomes of immunological assays in which they are used. Supernatant conditioned by growth of L929 cells has often been used to generate mouse macrophages from bone marrow in vitro but is subject to lot-to-lot variability. To reduce experimental variability and to standardize techniques across laboratories, we investigated recombinant M-CSF (rM-CSF) as an alternative supplement for BMDM maturation in the context of macrophage infection, using the intracellular bacterium Live Vaccine Strain (LVS) of Francisella tularensis as a prototype. We compared rM-CSF with L929 supernatant in terms of their effects on mouse and rat macrophage growth, maturation patterns, surface marker expression, and the expression of selected genes. Further, we compared macrophage infectivity and bacterial replication using LVS. Finally, we compared the in vitro function of BMDMs co-cultured with splenocytes from vaccinated animals in terms of their control of intramacrophage bacterial replication, as well as production of cytokines and nitric oxide. We demonstrated that rM-CSF produced BMDMs with similar, or minimal, phenotypic and gene expression outcomes compared to those generated with media containing L929 supernatant. Most importantly, functional outcomes were similar. Taken together, our data support the use of the rM-CSF in cell culture media as an alternative to L929-supplemented media for functional bioassays that use C57BL/6J mouse or Fischer 344 rat BMDMs to study intracellular infections. This comparison therefore facilitates future protocol standardization.

中文翻译：

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rM-CSF可有效替代L929，以产生小鼠和大鼠骨髓来源的巨噬细胞，以进行针对细胞内细菌免疫的体外功能研究。

用于制备骨髓源巨噬细胞（BMDM）的方法可能会影响使用它们的免疫测定的结果。由L929细胞生长调节的上清液通常用于体外从骨髓中产生小鼠巨噬细胞，但批次间存在差异。为了减少实验的可变性并使实验室之间的技术标准化，我们使用巨细胞弗朗西斯菌（Francisella tularensis）的细胞内细菌活疫苗株（LVS）作为巨噬细胞感染的背景，研究了重组M-CSF（rM-CSF）作为BMDM成熟的替代补充剂。一个原型。我们将rM-CSF与L929上清液对小鼠和大鼠巨噬细胞生长，成熟模式，表面标志物表达以及所选基因的表达进行了比较。进一步，我们比较了使用LVS的巨噬细胞感染性和细菌复制。最后，我们比较了与已接种动物脾细胞共培养的BMDM的体外功能，它们对巨噬细胞内细菌复制的控制以及细胞因子和一氧化氮的产生。我们证明，与用含有L929上清液的培养基产生的BMDM相比，rM-CSF产生的BMDM具有相似或最小的表型和基因表达结果。最重要的是，功能结局相似。综上所述，我们的数据支持在细胞培养基中使用rM-CSF替代L929补充的培养基，用于使用C57BL / 6J小鼠或Fischer 344大鼠BMDM研究细胞内感染的功能性生物测定。因此，该比较有助于将来的协议标准化。