many salutary functions in the heart, and may be critical for normal cardiac homeostasis. Materials and Methods Mice Adult Cx3cr1GFP/+ transgenic mice were a gift from C. Gross (European Molecular Biology Laboratory, Monterotondo, Italy). All mice used were in the C57BL/6 background; they were maintained in a specific pathogen-free(SPF) environment and fed standard mouse diet ad libitum. All procedures conducted on mice were approved by the respective animal ethics committees. Mouse procedures conducted are as described in ethics applications MARP/2010/017 and MARP/2011/014 approved by the Monash Animal Research Platform 2 (MARP2) Animal Ethics Committee. Preparation of single cell suspensions from different tissues Prior to isolation of cells from all tissues, except from the peritoneal cavity, mice were perfused with ice-cold Hank’s Balanced Saline Solution (HBSS), through the left ventricle to clear blood. For preparing single cell suspensions from hearts, kidneys and lungs, tissues were isolated, placed in HBSS solution on ice before being finely minced with surgical scissors and incubated in collagenase type II (Worthington Laboratories)/ HBSS enzyme solution for 30 minutes at 37uC with gentle agitation. Dissociated cells and undigested tissue were centrifuged and resuspended in collagenase-dispase (Roche Applied Science)/ HBSS enzyme solution, and incubated for a further 20 min at 37uC with gentle shaking. Cell/tissue suspensions were filtered through 70 mm filters, centrifuged and resuspended in 2% foetal bovine serum (FBS)/PBS until further analysis. Single cell suspensions from adipose were prepared as described above with the omission of the tissue digestion in collagenase-dispase/HBSS enzyme solution. The adipose tissue cell suspensions were centrifuged after the collagenase type II/HBSS solution in- cubation floating fatty debris was discarded, and cell pellets resuspended in 2% FBS/PBS, before analysis. Splenic single cell suspensions were prepared by mashing spleens against a 70 mm filter in 2% FBS/PBS using a syringe plunger, and the isolated cell suspensions passed through more 70 mm filters. Red blood cell lysis was conducted with splenic cell suspensions using Red Blood Cell Lysis Solution (10 mM KHCO3, 150 mM NH4CL, 0.1 mM EDTA pH 8.0). Peritoneal cavity cells were isolated by lavage of mouse peritoneal cavities with Dulbecco’s Modified Eagle’s Medium (DMEM) and centrifugation of the resulting cell suspensions. Mouse brain single cell suspensions were prepared by mincing tissue with a scalpel and incubating them in collagenase-dispase/HBSS solution supplemented with 0.025 U/ ml DNaseI (Roche Applied Science) for 30 min at 37uC with gentle agitation. Cell/tissue suspensions were filtered through 70 mm filters and centrifuged (5 min at 4uC, 300 g). Resulting pellets were resuspended in 37% Percoll solution, and separated by gradient centrifugation by placing cell suspension on top of a dense 70% Percoll solution before centrifugation for 25 min at 4uC. Cells accumulated at the interphase of the two solutions were carefully collected and washed in 2% FBS/HBSS before analysis. Macrophages in the Adult Murine Heart rmC5-3, BD Pharmingen), CD11c (clone HL3, BD Pharmingen), CD86 (clone GL1, BD Pharmingen), NK1.1 (clone PK136, BD Pharmingen), B220 (clone RA3-6B2, BD Pharmingen), CD3e (clone 145-2C11, BD Pharmingen), LY6C/G (clone RB6-8C5, Biolegend), Mrc1 (CD206), (clone MR5D4, Biolegend), CD45 (clone 30-F11, Biolegend), CD11b (clone M1/70, Biolegend), CD16/CD32 (clone 93, eBioscience), GFP (Chicken Polyclonal (ab13970), Abcam), Lyve-1 (Rabbit Polyclonal (ab14917), Abcam), CD163 (Rabbit Polyclonal (M-96), Santa Cruz Biotechnology). Dextran uptake was detected by flow cytometry in cardiac single cell suspensions after intravenously injecting mice with 50 mg tetramethylrhodamine-labelled 2 MDa dextran (Invitrogen) 20 hours before cell isolation. Cells were analysed by a FACS- Canto cytometer (Becton Dickinson) using FlowJo Software. Fluorescence activated cell sorting (FACS) was conducted using FACS-ARIA cell sorter (Becton Dickinson). All sorted cells were isolated into DMEM supplemented with 10% FBS. Quantitative Real Time PCR (qRT-PCR) Total RNA was isolated from FACS-sorted CX3CR1 positive cells using RNeasy Mini Kit (QIAGEN). DNaseI treatment and cDNA synthesis was performed using QuantiTect Reverse Tran- scription Kit (QIAGEN). TaqMan gene expression assays (Applied Biosystems) were used for quantification of the mRNA levels of the genes of interest. Probes for TaqMan Gene Expression Assays are as listed: IL6 (Mm00446190_m1), IL10 (Mm00439614_m1), IL1b (Mm01336189_m1), IGF1 (Mm00439560_M1), Lyve1 (Mm00475056_m1), CXCL1 (Mm00433859_m1), CXCL2 (Mm00436450_m1), Chi3l3 (Mm04213363_u1), TIMP2 (Mm00441825_m1), MMP13 (Mm00439491_m1), Stab1 (Mm00460390_m1), Arg1), (Mm00475988_m1), Hmox1 (Mm00516005_m1), Angpt1 (Mm00456503_m1), Rps18 (Mm02601777_g1), Mouse ACTB (actin, beta), Endogenous Control (4352341E). TaqMan assays for bActin (VIC) or Rps18 (FAM) served as endogenous controls. Data was generated with the Applied Biosystems 7500 Real-Time PCR System. Data analysis was performed using the DDC(T) method. Immunostaining and confocal microscopic analysis For preparation of heart tissue for immunostaining, animals were perfused with fresh ice-cold 4% formaldehyde/PBS through the left ventricle and tissue was harvested and incubated in fresh 4% formaldehyde/PBS overnight. For thick section staining, tissue was sectioned using a vibrating blade microtome (Leica Micro- systems). Prepared sections were permeabilised with 0.2% Triton X-100 (Sigma)/PBS solutions, before blocking in 1% goat-serum/ 0.2% Triton X-100/PBS. Standard immunostaining protocols were used for following steps. The primary antibodies used for staining of sections are summarised above. Biotin-conjugated isolectin B4 (Vector Laboratories) was used to stain vasculature. Alexa Fluor conjugated secondary antibodies (Invitrogen) and streptavidin (Invitrogen) were used for fluorescence staining. Fluorescent microscopy images were obtained using Leica SP5 confocal laser scanning microscope (Leica Microsystems) and Nikon C1 confocal laser scanning microscope (Nikon Instruments). Two and three-dimensional images were prepared using Imaris software (Bitplane), with adjustments made to brightness and contrast. Flow cytometry and antibodies used The antibodies used for staining of single cell suspensions after FC receptor blocking with CD16/CD32, and for immunofluores- cence microscopy are as listed: F4/80 (clone BM8, eBiocience), MHC-II (IA-b) (clone AF6-120.1, BD Pharmingen), CD14 (clone Microarray analysis Expression analysis was conducted on FACS sorted CD45+CD11b+GFP+ cells from the mouse heart, spleen and brain. Total RNA samples were isolated from three independent biological replicates using the RNeasy Mini kit (Qiagen), and were PLoS ONE | www.plosone.org 2 May 2012 | Volume 7 | Issue 5 | e36814