Macrophages in the Adult Murine Heart Figure 1. Detection of GFP+ cells within adult Cx3cr1GFP/+ mouse hearts. (A) Scatter profiles showing CD45+ cells in the adult mouse heart (left panel) and the expression of CD11b and GFP within this population (right panel). (B) Scatter profile of GFP+ cells (green), with CD45+GFP2 (black) and CD452GFP2 cells and debris (grey) shown (top left panel). (Top right panel) scatter profile of CD45+ cells (as shown in figure 1A left panel) with GFP+ cells, GFP2 cells and debris indicated (green, black and grey respectively; based on gating from figure 1A). (Bottom left panel) scatter profile of CD11b and CD45 expression within GFP+ cells. (C) 2 MDa dextran uptake by CD45+CD11b+GFP+ cells. (D) 45 mm projection view of a cross sectional image of the adult Cx3cr1GFP/+ mouse heart left ventricle. Scale bar = 150 mm. (E) 3D projections of typical GFP+ (green) cells from the left ventricular myocardium with isosurface rendering of nuclei (blue). Figures representative of at least 3 independent experiments. (F) Magnified projection view of GFP+ cells with perivascular position shown (capillaries stained with IB4; nuclei stained with DAPI). Scale bar = 5 mm. (G) Optical section of GFP+ cells with perivascular (capillaries stained with IB4) and pericardiomyocyte position (cardiomyocyte cell surface stained with WGA; nuclei stained with DAPI). Scale bar = 10 mm. All images representative of at least 3 similar independent experiments. doi:10.1371/journal.pone.0036814.g001 In further comparative analyses, 21 probe sets were identified as 20-fold or greater enriched (p#0.05) in both cTMs and GFP+ cells from the brain, in comparison to GFP+ cells from the spleen (data not shown). Notably, these included components of complement pathway, macrophage related genes Stab1 and PMP22 [18,19]. Conversely, 2 probe sets (Bex6, Apol7c) were identified as enriched 20-fold or more (p#0.05) in GFP+ cells from the spleen compared to GFP+ cells from the heart or brain. Gene expression patterns identified by gene array analysis were validated by quantitative reverse transcriptase-PCR (qRT-PCR), confocal fluorescence microscopy and flow cytometric analysis. For validation of gene expression by qRT-PCR (Fig. 3 c), we chose probes enriched in cTMs (Lyve-1, IGF1, MMP13, CXCL1, CXCL2, IL1b, and IL6), enriched in the splenic GFP+ cells (Angpt1), or enriched in both cTMs and brain GFP+ cells (Timp2, Stab1, Hmox1). qRT-PCR confirmed gene expression patterns observed in microarray analysis, however, levels of enrichment determined by qRT-PCR and gene array analysis varied. Expression of Mrc1, CD163, and Lyve-1 was validated by confocal fluorescence microscopy of adult heart sections from Cx3cr1GFP/+ mice (Fig. 4 c). In addition, Mrc1 expression was also confirmed by flow cytometric analysis (Fig. 4 d). PLoS ONE | www.plosone.org 4 May 2012 | Volume 7 | Issue 5 | e36814