Laboratory for Biological Mass Spectrometry, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK.
Laboratory for Biological Mass Spectrometry, Biosciences Institute, Newcastle University, Newcastle upon Tyne, UK
Life Sci Alliance. 2021 Apr 14;4(6). doi: 10.26508/lsa.202000957. Print 2021 Jun.
BMDMs are a key model system to study macrophage biology in vitro. Commonly used methods to differentiate macrophages from BM are treatment with either recombinant M-CSF or the supernatant of L929 cells, which secrete M-CSF. However, little is known about the composition of L929 cell-conditioned media (LCCM) and how it affects the BMDM phenotype. Here, we used quantitative mass spectrometry to characterise the kinetics of protein secretion from L929 cells over a 2-wk period, identifying 2,193 proteins. Whereas M-CSF is very abundant in LCCM, we identified several other immune-regulatory proteins such as macrophage migration inhibitory factor (MIF), osteopontin, and chemokines such as Ccl2 and Ccl7 at surprisingly high abundance levels. We therefore further characterised the proteomes of BMDMs after differentiation with M-CSF, M-CSF + MIF, or LCCM, respectively. Interestingly, macrophages differentiated with LCCM induced a stronger anti-inflammatory M1 phenotype that those differentiated with M-CSF. This resource will be valuable to all researchers using LCCM for the differentiation of BMDMs.
BMDMs 是体外研究巨噬细胞生物学的重要模型系统。常用的将 BM 中的巨噬细胞分化的方法是用重组 M-CSF 或分泌 M-CSF 的 L929 细胞上清液处理。然而，关于 L929 细胞条件培养基（LCCM）的组成以及它如何影响 BMDM 表型的了解甚少。在这里，我们使用定量质谱法在 2 周的时间内表征 L929 细胞分泌蛋白的动力学，鉴定出 2193 种蛋白质。尽管 LCCM 中 M-CSF 非常丰富，但我们鉴定出其他几种免疫调节蛋白，如巨噬细胞移动抑制因子（MIF）、骨桥蛋白和趋化因子如 Ccl2 和 Ccl7，其丰度水平高得惊人。因此，我们分别用 M-CSF、M-CSF+MIF 或 LCCM 进一步对 BMDM 分化后的蛋白质组进行了表征。有趣的是，用 LCCM 分化的巨噬细胞诱导出比用 M-CSF 分化的更强的抗炎 M1 表型。这个资源将对所有使用 LCCM 来分化 BMDM 的研究人员都很有价值。
