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. Author manuscript; available in PMC: 2014 Jul 1.
Published in final edited form as: Nat Neurosci. 2013 Dec 8;17(1):131–143. doi: 10.1038/nn.3599

Figure 6. Role of TGF-β in the development of microglia in vitro.

Figure 6

(a and b) MG400 analysis of (a) upregulated microglial genes and (b) downregulated microglial genes in cultured adult microglia in the presence of MCSF and TGF-β1 in comparison to adult microglia cultured in the presence of GM-CSF or MCSF alone (see Source data – Figure 6). One representative of three individual experiments is shown. (c) qPCR analysis of 6 selected microglial genes in cultured adult microglia in the presence of MCSF and TGF-β1. Gene expression level was normalized against Gapdh using ΔCt (n = 6, each biological triplicate consisted of two wells per treatment). Bars show mean normalized intensity ± s.e.m. *P<0.05, **P<0.01, ***P<0.001, F2,15=5.829; 1-Way ANOVA followed by Dunnett’s multiple-comparison post-hoc test. (d) Principal component analysis (PCA) of different cell populations based on MG400 expression profile. (e and f) qPCR analysis of Fcrls, Sall1 and Gpr-34 gene expression (e) and miR-99a, miR-125b-5p and miR-342-3p expression (f) in RAW264.7 macrophages, N9 microglia cell line and adult microglia cultures. Cells were untreated or cultured in the presence of MCSF, GM-CSF, or polarized for 48h to M0 (MCSF+TGF-β1), M1 (GM-CSF+IFNγ+LPS) or M2 (MCSF+IL4) phenotypes. RAW264.7 macrophages and N9 cells survive without MCSF or GM-CFS, whereas adult microglia require either MCSF or GM-CSF. Gene expression level was normalized against Gapdh using ΔCt (n = 3). miRNA expression level was normalized against U6 miRNA using ΔCt (n = 3). Bars show mean ± s.e.m. Shown is one of two individual experiments.