analysed with a Bioanalyzer (Agilent Technologies) system for RNA quality and further processed at Genecore (EMBL Heidelberg, Germany). Microarray experiments were performed on Affymetrix Gene- chipH HT Mouse Genome 430.2 Array Plate Sets (Agilent Technologies). Data were extracted and normalised using Gene- Spring GX 10.0 software (Build 77727; Agilent Technologies and Strand Life Sciences). Probes were filtered to include those where at least 1 of 3 samples had a normalised intensity value between 3.0 and 13.96. Each dataset was derived from three biologically independent replicate samples. In comparative analyses probes were filtered to include those with p#0.05 (T-test with Bonferroni adjustment). All microarray data files are available in the ArrayExpress database (www.ebi.ac.uk/arrayexpress) under ac- cession number E-MEXP-3347. Statistical analysis For qRT-PCR experiments, student’s t-test (two tailed, assum- ing normal distribution) was applied using Microsoft Excel software where respective genes from various tissues were tested for significance against values determined from the heart. Differences were considered statistically significant where p#0.05. Values displayed in histograms represent mean 6 SEM. Results GFP+ cells within Cx3cr1GFP/+ knock-in mouse hearts are myeloid cells To characterise haematopoietic cells, and more precisely myeloid cells, in the adult murine myocardium, we examined GFP+ cells extracted from the adult myocardium of Cx3cr1GFP/+ knock-in transgenic mice [3]. Flow cytometry analysis of cells isolated from adult Cx3cr1GFP/+ mouse hearts identified a large haematopoietic (CD45+) and GFP+ population (Fig. 1 a). Although no clear stratification of GFPhi and GFPlo cells was clearly visible, both GFPhi and GFPlo cells (referred to as GFP+) were gated together for subsequent analyses to avoid bias towards over representing data from GFPhi cells. The majority of CD45+ cells were GFP+, most of which were myeloid as identified by CD11b staining (Fig. 1 a). Using an alternative gating approach, we confirmed that GFP+ cells from adult Cx3cr1GFP/+ mouse hearts were CD45+ and CD11b+, and that GFP+ cells represent a distinct population in scatter profiles of CD45+ cells (Fig. 1 b). To assess macropinocytosis, a functional hallmark of mononuclear phago- cytes [13], we injected Cx3cr1GFP/+ mice with TRITC-labelled 2 MDa dextran, which was macropinocytosed by cardiac GFP+ cells (Fig. 1 c). Confocal fluorescence microscopy of the myocardium revealed stellate shaped GFP+ cells distributed throughout the heart (Fig. 1 d). These cells are mononuclear and typically have a small cell body with one or two cytoplasmic/dendritic projections (Fig. 1 e). The morphology and mononuclear nature of these cells rules out the possibility that they are granulocytes or neutrophils, whereas their extensive macropinocytosis reinforces a cardiac tissue macrophage (cTM) phenotype. cTMs are in direct contact with vascular endothelial cells and cardiomyocytes To determine the anatomical position of cTMs within the adult Cx3cr1GFP/+ mouse myocardium in relation to the coronary vasculature, confocal fluorescence microscopy was conducted after isolectin B4 (IB4) staining, which labels endothelial cells. IB4 has previously been shown to stain macrophages [14] and Cx3cr1 expression has been found in endothelial cells of the skin [15]. However, in our experiments we did not observe IB4 staining in Macrophages in the Adult Murine Heart cTMs nor did we detect any GFP+ endothelial cells. cTMs maintained a perivascular position, associating with at least one capillary endothelial cell (Fig. 1 f, g), To determine the position of cTMs in relation to cardiomyocytes, tissue sections were stained with the lectin wheat germ agglutinin (WGA), revealing that cTMs were positioned in direct cell contact with cardiomyocytes (Fig. 1 g). Immunophenotype of CD45+ cells in the hearts of adult Cx3cr1GFP/+ knock-in mice Cell surface marker analysis further distinguished cTMs (CD45+CD11b+GFP+) from the CD45+ non-myeloid population (CD45+CD11b2GFP2) (Fig. 2). The cTM population stained positive for macrophage marker F4/80, lipo-polysaccharide re- ceptor CD14, and maturation/activation markers MHC-II (IAb) and CD86. Low or no staining was detected for CD11c, natural killer cell marker NK 1.1, and lymphocyte markers B220 and CD3e. These data indicate that cTMs within the adult mouse myocardium are macrophages. In contrast, the cardiac non- myeloid cell population stained for B220 and CD3e, and a small subset stained positive for NK 1.1, indicating they comprise a lymphoid population and natural killer cells. In addition, these cells stained positive for MHC-II, consistent with the presence of B-cells in this subset, and did not stain for F4/80, CD86, CD14, or CD11c (Fig. 2). cTMs have a unique gene expression signature compared to GFP+ cells in the spleen and brain To develop a gene signature for cTMs, we conducted fluorescent activated cell sorting (FACS) of CD45+CD11b+GFP+ cells from adult Cx3cr1GFP/+ mouse hearts (cTMs), spleens and brains and conducted Affymetrix gene array analysis. GFP+ cells from adult Cx3cr1GFP/+ mouse spleens are comprised of monocytes, macrophages and DCs [16], whereas TMs (microglia) comprise GFP+ cells from the brain [17]. Comparison of normalised gene expression between cTMs and GFP+ cells from the spleen and brain revealed common features including similar expression levels for housekeeping genes, GAPDH, b-tubulin 1, b-actin and macrophage-specific markers, colony stimulating factor 1 receptor (CSF1-r) and CD68 (Fig. SI), confirming the macrophage phenotype of cTMs. Analysis of genes expressed from each cell type (defined as probes expressed above a normalised value of 4) showed 25,532 probes expressed by all three GFP+ populations (Fig. 3 a). cTMs also expressed 509 unique probes that were not expressed in GFP+ cells in the spleen or brain (Fig. 3 a). Conversely, splenic GFP+ cells expressed 424 unique probes and brain GFP+ cells expressed 1059 unique probes (Fig. 3 a). Together, these data underscore the heterogeneity of these three myeloid cell populations. To identify genes that are highly enriched in cTMs, compared to both splenic and brain derived GFP+ cells, we filtered probe sets that were 20-fold or more enriched in cTMs (p#0.05). The filter isolated 42 probes comprising 35 genes (Fig. 3 b, and Table S1). Of these, at least 12 genes (Lyve-1, Cxcl1, Mmp13, Cxcl2, Pf4, Cd163, Cxcl3, Il10, Tnfsf9, Mrc1, Vsig4, and Colec12) have previously-identified immunological functions. Greater than 100- fold enrichment was observed in cTMs for the genes Lyve-1, Cxcl1, Mmp13, Cxcl2, Cbr1, CD163 and Emp1. Highly expressed cardiomyocyte structural genes were also detected (Mylk, Actc1, Tnnt2, Myl3 and Myh6). Although cardiomyocyte genes were only moderately enriched, the presence of cardiomyo- cyte transcripts indicates a degree of contamination in cTM RNA samples. PLoS ONE | www.plosone.org 3 May 2012 | Volume 7 | Issue 5 | e36814