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 Apoe2/2 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). EAE induction At 12–24 weeks of age, EAE was induced in male and female mice weighing less than 32 g as described [26] and monitored for signs of disease as described [35]: 0, no disease; 1, decreased tail tone and/or poor righting ability; 2, abnormal gait and/or hind limb weakness; 2.5, partial hind limb paralysis; 3, complete hind limb paralysis; 3.5, ascending paralysis; 4, tetraplegia; 5, death. Mice were sacrificed at a score of 3–4. Microscopy Brains and spinal cords were either perfused with phosphate- buffered saline (PBS) and frozen whole in OCT or perfused with or 4% PFA and processed into free-floating 30-mm sections, as described [26]. Sections were stained for CD45, as described [44]. For details on histological methods, please see Text S1. To visualize the distribution of cells expressing GFP (CX3CR1) and RFP (CCR2), free-floating sections were stained with 15 nM DAPI for 5 min, and whole-brain sections were scanned using a Leica DM5500B with motorized x,y stage. High-power confocal images are shown as projections of 7–10-mm z stacks at 40x magnification and scanned at a step size of 1–1.5 mm. In some experiments, brain sections were incubated overnight at 4uC with 7–4 antibody (Cedarlane, Ontario, Canada), washed, and incubated at room temperature for 2 h with biotinylated-rabbit anti rat secondary antibody (Jackson ImmunoResearch). After additional washes, tissues were incubated at room temperature for 1 h with strepatvidin-Cy5 conjugated, rinsed and mounted in Fluorsave reagent (Calbiochem). Tissues were imaged at 40x magnification on a Zeiss 510 confocal microscope. Flow cytometry For flow cytometric analysis of CNS tissues, mice were perfused with cold Hank’s balanced salt solution. The brains were dissected and the spinal cords were flushed with 0.5 ml of cold PBS. Tissues were homogenized with Dounce homogenizers, and cells were separated in a discontinuous 30/70 Percoll gradient [45]. Cells were collected from the 30/70 interphase and washed in PBS. Blood was collected by cardiac puncture of anesthetized mice, depleted of red blood cells in lysis buffer (0.15 mol/l NH4Cl, 1 mmol/l KHCO3, 0.1 mmol/l EDTA), and washed in staining buffer (PBS/0.1% BSA). For flow details, please see Text S1. Sterile peritonitis model Peritonitis was induced with thioglycollate as described [25]. Peritoneal leukocytes were collected 24 h later by flushing the peritoneal cavity with 10 ml of PBS. Peritoneal cells were washed with staining buffer and stained for F4/80 and 7-4 as described above. Novel CCR2-RFP Mice in EAE Quantitative RT-PCR RNA was isolated from FACS-sorted cell populations with the Arcturus PicoPure kit (Molecular Devices, Sunnyvale, CA) according to the manufacturer’s instructions. Quantity was assessed with a Nanodrop 1000, and RNA was reverse transcribed with TaqMan reverse-transcription reagents. Quan- titative real-time PCR was carried out with TaqMan PCR Master Mix, TaqMan gene expression assays, and an Applied Biosystems 7900HT thermal cycler. All TaqMan reagents were from Applied Biosystems. Reactions were run in quadruplicate, and CCR2 and RFP mRNA expression levels were normalized to beta-actin. Statistical analysis Data are presented as mean 6 SEM. Differences between Cx3cr1+/2Ccr2+/2 and Cx3cr1+/2Ccr22/2 mice were analyzed with a two-tailed nonparametric Mann-Whitney test. Supporting Information Text S1 Supporting Materials and Methods. Found at: doi:10.1371/journal.pone.0013693.s001 (0.03 DOC) MB Table S1 Primers used for tail-DNA genotyping. *Primers A-B amplify the wild type allele, whereas primers B-C identify the knockout allele. Found at: doi:10.1371/journal.pone.0013693.s002 (0.03 MB DOC) Figure S1 Analyses of cytoplasmic CCR2. WT, Ccr2RFP/+, and Ccr2RFP/RFP mice were bred onto the Apoe2/2 background and fed a high-fat diet for 8 weeks. Intact and permeabilized monocytes, identified by CD115 staining, were stained for CCR2 and Ly6C. Boxes indicate demarcation of monocytes into Ly6Chi and Ly6Clo populations. Dashed line indicates the cutoff for positive CCR2 staining, using Ccr2RFP/ RFP cells as the negative control. Permeabilization did not increase background staining in the CCR2 knockout mice (not shown). Results are representative of four experiments. Found at: doi:10.1371/journal.pone.0013693.s003 (0.50 MB EPS) Figure S2 Analysis of spinal inflammation in CCR2-deficient mice. Spinal cord tissues were fixed and stained with anti-CD45 antibodies to compare the distribution of inflammatory cells in double heterozygous Ccr2RFP/+ Cx3cr1GFP/+ mice (A,B) and Ccr2RFP/RFP Cx3cr1GFP/+ mice (C,D) at peak EAE disease. The extent of disease was similar in both genotypes. WM: white matter. GM: gray matter. Found at: doi:10.1371/journal.pone.0013693.s004 (1.97 MB EPS) Acknowledgments We thank Dr. Littman for the generous gift of the CX3CR1-GFP mice, and Jenny Garcia for 7/4 staining, and Dr. Colleen Witt for assistance with confocal microscopy. We thank Stephen Ordway for editorial assistance, Daryl Jones for manuscript preparation, and John Carroll for preparation of the figures. Author Contributions Conceived and designed the experiments: NS AC RMR IFC. Performed the experiments: NS AC KC MM AC CLT. Analyzed the data: NS AC RMR IFC. Wrote the paper: NS AC RMR IFC. PLoS ONE | www.plosone.org 11 October 2010 | Volume 5 | Issue 10 | e13693