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 CD45^low expressing cells (microglia; red; CD11b + CD45^low). Spleen monocytes/macrophages were first gated based on CD11b⁺ and CD45^high expression, followed by gating for Ly6G^low and Ly6C^high expression (spleen monocytes/macrophages; blue; CD11b + CD45^low Ly6G^low Ly6C^high). 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⁺ CD45^low) and spleen monocytes/macrophages (CD11b⁺ CD45^high Ly6G^low Ly6C^high) by quantitative RT-PCR. (b) Microglia and spleen monocytes were freshly isolated from 8 to 12 weeks old male Cx3cr1^GFP/WT; Ccr2^RFP/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⁺ CD45^high Ly6G^low Ly6C^high or RFP⁺GFP⁺ cells; blue; n = 3)