Generation and Characterization of CCR2-RFP and CX3CR1-GFP Mice

CCR2^RFP mice were created as described [25] (link) (see Figure 1 and Text S1). Founder mice were crossed with Cre-deleter mice [43] (link) to remove the neo gene and backcrossed onto the C57Bl/6 line nine times. To generate homozygous Ccr2^RFP/RFPCx3cr1^GFP/GFP mice, we crossed Ccr2^RFP/RFP with Cx3cr1^GFP/GFP C57Bl/6 mice (a gift of D.R. Littman), and the progeny were backcrossed onto C57Bl/6. Mice that had undergone chromosome recombination between the CCR2 and CX3CR1 loci were selected by being positive for both RFP and GFP by flow cytometry of tail vein blood.
Unless stated otherwise, all mice were backcrossed seven to nine times on C57Bl/6 and were 2–6 months of age at sacrifice. Some mice were crossed with C57Bl/6 Apoe^−/− mice. These mice were fed a Western diet (42% of calories from fat) (Harlan Teklad, TD88137) for 8 weeks, starting at 6–8 weeks of age. All other mice were fed standard chow. Mice were bred at the Gladstone Institutes and the Biological Resources Unit, Cleveland Clinic, Lerner Research Institute. Animal experiments were performed according to the protocols approved by the Institutional Animal Care and Use Committee at the Cleveland Clinic, UCSF, and UTSA following the National Institute of Health guidelines for animal care. Mice were genotyped by PCR using tail DNA, and chemokine receptor–specific primers (Invitrogen, Carlsbad,CA) (Table S1).