Expression of CCR2 and RFP in sorted cell populations.

(A) CD115⁺ monocytes were sorted by flow cytometry into populations that did or did not express cell-surface CCR2. Total RNA was isolated, pooled from three mice per genotype, and analyzed for CCR2 mRNA and RFP mRNA by quantitative RT-PCR. Probe specificity was confirmed with WT and Ccr2^−/− cells. Expression levels were normalized to beta-actin, and the results are expressed relative to the concentration of CCR2 mRNA in monocytes expressing cell-surface CCR2. (B) NK and T cells were sorted by flow cytometry into RFP⁺ and RFP^– fractions. Expression of CCR2 and RFP mRNA was characterized as in (A) and normalized to the amount of CCR2 RNA in the RFP⁺ T-cell fraction from Ccr2^+/RFP mice. (C to G) WT, Ccr2^RFP/+, and Ccr2^RFP/RFP mice were bred onto the Apoe^−/− background and fed a high-fat diet for 8 weeks. Peripheral blood monocytes were stained for CD115, CCR2, and Ly6C and analyzed by flow cytometry. (C) Gating on monocytes. (D) Ly6C and RFP expression of monocytes. Dashed line indicates the cutoff for a positive RFP signal. (E) Mean RFP fluorescence intensity in Ly6C^hi and Ly6C^lo subsets. Bars indicate SEM. n = 4 mice/group. P<0.008. (F) CCR2 and RFP expression of Ly6C^hi monocytes. (G) CCR2 and RFP expression in Ly6C^lo monocytes. CCR2-positive and CCR2-negative gates were based on CCR2-deficient Ccr2^RFP/RFP mice. (H to K) Flow cytometric analysis of Ly6C, RFP, GFP and cell-surface CCR2 expression on monocytes from Ccr2^RFP/+Cx3cr1^GFP/+ mice. CD115+ monocytes were gated (H) by Ly6C/GFP expression and analyzed for RFP (I) and CCR2 (J) expression. Ccr2^RFP/RFPCx3cr1^GFP/+ monocytes (K) were used as a negative control for CCR2 surface-staining. Results are representative of four mice in two similar experiments.