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Fig. 3 | Acta Neuropathologica Communications

Fig. 3

From: Comprehensive gene expression meta-analysis identifies signature genes that distinguish microglia from peripheral monocytes/macrophages in health and glioma

Fig. 3

Validation of SGmic and SGmac genes by quantitative RT-PCR analysis in microglia and spleen monocytes/macrophages freshly isolated from two different mouse models. (a) Microglia and spleen monocytes/macrophages were freshly isolated from 12-weeks old male C57BL/6J WT mice by FACS. Microglia were first gated as CD11b+ cells against forward scatter (FSC) and subsequently selected as CD45low expressing cells (microglia; red; CD11b + CD45low). Spleen monocytes/macrophages were first gated based on CD11b+ and CD45high expression, followed by gating for Ly6Glow and Ly6Chigh expression (spleen monocytes/macrophages; blue; CD11b + CD45low Ly6Glow Ly6Chigh). Expression of SGmic (P2ry13, P2ry12, Gpr34, Slc2a5, Siglec-H, Olfml3, Tmem119, and Fcrls) and SGmac (F10, Emilin2, F5, C3, Gda, Mki67, Sell, Hp) genes was assessed in microglia (CD11b+ CD45low) and spleen monocytes/macrophages (CD11b+ CD45high Ly6Glow Ly6Chigh) by quantitative RT-PCR. (b) Microglia and spleen monocytes were freshly isolated from 8 to 12 weeks old male Cx3cr1GFP/WT; Ccr2RFP/WT mice by FACS. Microglia were gated as GFP-expressing cells against FSC (microglia; red; GFP+RFP). Spleen monocytes/macrophages were isolated as RFP-expressing cells and sorted as two populations based on their GFP-expression levels as RFP+GFP+ (spleen monocytes/macrophages; blue) and RFP+GFP cells (spleen monocytes/macrophages; purple). Expression of SGmic (P2ry13, P2ry12, Gpr34, Slc2a5, Siglec-H, Olfml3, Tmem119, and Fcrls) and SGmac (F10, Emilin2, F5, C3, Gda, Mki67, Sell, Hp) genes was assessed in microglia (GFP+RFP cells) and spleen monocytes/macrophages (RFP+GFP+ cells) by quantitative RT-PCR. Bar graphs represent the log fold change expression of each gene normalized to Hprt and in the isolated microglia population relative to the peripheral monocytes/macrophage population (CD11b+ CD45high Ly6Glow Ly6Chigh or RFP+GFP+ cells; blue; n = 3)

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