www.sciencemag.org/cgi/content/full/325/5940/612/DC1

Supporting Online Material for

Identification of Splenic Reservoir Monocytes and Their Deployment to Inflammatory Sites

Filip K. Swirski,* Matthias Nahrendorf, Martin Etzrodt, Moritz Wildgruber, Virna Cortez-Retamozo, Peter Panizzi, Jose-Luiz Figueiredo, Rainer Kohler, Aleksey Chudnovskiy, Peter Waterman, Elena Aikawa, Thorsten R. Mempel,

Peter Libby, Ralph Weissleder,* Mikael J. Pittet*

*To whom correspondence should be addressed. E-mail: fswirski@mgh.harvard.edu (F.K.S.), rweissleder@mgh.harvard.edu (R.W.), or mpittet@mgh.harvard.edu (M.J.P.)

Published 31 July 2009, Science 325, 612 (2009)

DOI: 10.1126/science.1175202

This PDF file includes

Materials and Methods Figs. S1 to S18 Tables S1 to S4 References

Other Supporting Online Material for this manuscript includes the following: (available at www.sciencemag.org/cgi/content/full/325/5940/612/DC1)

Movies S1 to S10

SUPPORTING ONLINE MATERIAL

Materials and MethodsAnimals. C57BL/6J, B6.129P-Cx3cr1tm1Litt/J (Cx3cr1gfp/gfp), B6.129S4-Ccr2tm1Ifc/J (Ccr2–/–), B6.SJL-PtprcaPep3b/BoyJ (CD45.1+) and B6.129P2-Agtr1atm1Unc (At-1–/–) female mice (all from Jackson Laboratories) were used in this study. Cx3cr1gfp/+ mice were obtained by breeding Cx3cr1gfp/gfp mice with C57BL/6J mice. Cx3cr1gfp/+ mice have one Cx3cr1 allele replaced with cDNA encoding Egfp, and can be used to track monocytes (1). Mice were 8-12 weeks old, except Ccr2–/– which were 1 year old (spleens of young Ccr2–/– mice are very small). Female Wistar rats were ~230 g (from Jackson Laboratories).

Cells. Peripheral blood was drawn via cardiac puncture with citrate solution (100 mM Na-citrate, 130 mM glucose, pH 6.5) as anti-coagulant and mononuclear cells were purified by density centrifugation. Total blood leukocyte numbers were determined using acetic acid lysis solution (3% HEMA 3 Solution II, 94% ddH2O, 3% glacial acetic acid). After organ harvest, single cell suspensions were obtained from brain, gut, heart, kidney, liver, lung, muscle and pancreas by digestion with a cocktail of 450 U/ml collagenase I, 125 U/ml collagenase XI, 60 U/ml DNase I and 60 U/ml hyaluronidase (Sigma-Aldrich, St. Louis, MO) for 1 h at 37°C while shaking. Some spleens were also prepared with the digestion cocktail. Total viable cell numbers were determined using Trypan Blue (Cellgro, Mediatech, Inc, VA).

Flow Cytometry. Anti-CD90-PE, anti-CD90-FITC, 53-2.1 (BD Biosciences); anti-B220-PE, anti B220-FITC, RA3-6B2 (BD Biosciences); anti-CD49b-PE, anti CD49b-FITC, DX5 (BD Biosciences); anti-NK1.1-PE, anti-NK1.1-FITC, PK136 (BD Biosciences); anti-Ly-6G-PE, anti-Ly-6G-FITC, 1A8 (BD Biosciences); anti-CD11b-APC, M1/70 (BD Biosciences); anti-CD11b-PE (ED8) (Abcam); anti-CD11b-APC-Cy7 M1/70 (BD Biosciences); anti-F4/80-biotin, anti-F4/80-FITC, C1:A3-1 (BioLegend); anti-CD11c-biotin, anti-CD11c-FITC, anti-CD11c-APC, HL3 (BD Biosciences); anti-I-Ab-biotin, anti-I-Ab-FITC, AF6-120.1 (BD Biosciences); anti-Ly-6C-FITC, anti-Ly-6C-biotin, AL-21 (BD Biosciences); anti-CD43-FITC, S7 (BD Biosciences); anti-CD62L-FITC, MEL-14 (BD Biosciences); anti-CD68-FITC, FA-11 (AbD Serotec); anti-CD86-biotin, GL1 (BD Biosciences); anti-CD115-PE, 604B5-2E11 (AbD Serotec); anti-Mac-3-FITC, M3/84 (BD Biosciences); anti-Gr-1-PeCy7, RB6-8C5 (BD Biosciences); anti-CD45.2-FITC 104 (BD Biosciences); anti-CD45.1-biotin A20 (BD Biosciences); anti-CD45.1-APC A20 (BD Biosciences) were used for flow cytometric analyses in this study. Strep-PerCP (BD Biosciences) was used to label biotinylated antibodies. Monocytes were identified as CD11bhigh (CD90/B220/CD49b/NK1.1/Ly-6G)low (F4/80/I-Ab/CD11c)low

Ly-6Chigh/low. Macrophages/DCs were identified as CD11bhigh (CD90/B220/CD49b/NK1.1/Ly-6G)low (F4/80/I-Ab/CD11c)high Ly-6Clow or on the basis of F4/80 or CD11c expression only. Neutrophils were identified as CD11bhigh (CD90/B220/CD49b/NK1.1/Ly-6G)high (F4/80/I-Ab/CD11c)low Ly-6Cint. Monocyte and macrophage/DC numbers were calculated as total cells multiplied by percent cells within the monocyte/macrophage gate. Within this population, monocyte subsets were identified as (F4/80/I-Ab/CD11c)low and either Ly-6Chigh or Ly-6Clow. For calculation of total cell numbers in tissue, normalization to weight of tissue was performed. Data were acquired on an LSRII (BD Biosciences) and analyzed with FlowJo v.8.5.2 (Tree Star, Inc.). Cells were sorted on a BD FACSAria (BD Biosciences). For morphologic characterizations, sorted cells were prepared on slides by cytocentrifugation (Shandon, Inc.) at 10 g for 2 min, and stained with HEMA-3 (Fischer Scientific). For gene profiling studies, blood and spleens of 5 to 10 mice were pooled for each replicate. Splenic monocytes were enriched by lineage depletion using MACS LD columns (Miltenyi) and PE–conjugated antibodies against B220, CD49b, NK1.1, Ly-6G, CD90 and Ter-119 followed by anti-PE magnetic beads (Miltenyi). Lineage-depleted cells were further stained with specific antibodies to allow for phenotypic identification of monocyte subsets. Monocytes from blood were stained and sorted

without prior enrichment.

Microarray gene expression profiling. Monocyte subsets from blood and spleen of a group of four mice were isolated by fluorescence activated cell sorting (FACS) as CD11bhigh (CD90/B220/CD49b/NK1.1/Ly-6G)low (F4/80/I-Ab/CD11c)low Ly-6Chigh cells. To avoid effects of lengthy staining protocols on the transcriptome of the cells of interest we developed a protocol that allowed sorting of cell subsets into RNA lysis buffer within ~30 min after the animals were sacrificed. Briefly, heparin-blood was drawn from anesthetized mice by cardiac puncture and spleens were immediately homogenized through a nylon mesh into 3 ml of PBS. e volume of the heparinized blood was adjusted with PBS to 1 ml of blood and the antibody mix was added. Similarly the antibodies were added to 1 ml of the splenocyte suspension. Staining was performed for 10 min, samples were diluted by addition of 1 ml PBS, immediately loaded onto 2 ml histopaque, and spun for 10 min at 18 g, 22°C. e interphase was collected and diluted into one volume of sorting buffer containing PBS, 2% FCS and 2 mM EDTA. Cells were FACS-sorted without delay. e protocol neither imposed cell pelleting, extend dwelling on ice, nor induced major osmotic stress. Both preparations of blood and splenic monocytes were performed for each animal simultaneously and under the same conditions. Samples of 1,000 Ly-6Chigh blood and Ly-6Chigh splenic monocytes were collected directly into 20 µl lysis buffer of the PicoPure RNA isolation kit (Arcturus). Sorting times varied between 2 and 10 min. RNA extraction was subsequently performed according to the manufacturer’s instructions (Arcturus). RNA quality was assessed using RNA pico lab chips on the Agilent Bioanalyzer. For all samples a RIN above 8 could be achieved. On average 1,000 cells yielded 200 pg total RNA. All further steps were performed at the UCSF Shared Microarray Core Facilities according to standard protocols (http://www.arrays.ucsf.edu and http://www.agilent.com). RNA was amplified using the NuGen WT-Ovation Pico System, and the amplified cDNA was labeled using the FL-Ovation cDNA Fluorescent Module (NuGen Technologies, San Carlos, Ca). Briefly, input total RNA was reverse–transcribed into cDNA and then amplified using a linear isothermal amplification process (SPIA). e amplified products were CY-3 labeled and fragmented according to manufacturer’s guidelines. Labeled cDNA was assessed using the Nanodrop ND-100 (Nanodrop Technologies, Inc., Wilmington DE) and the Agilent 2100 Bioanalyzer; equal amounts of Cy3-labeled target were hybridized to Agilent whole mouse genome 4x44K Ink-jet arrays. Hybridizations were performed for 14 h according to the manufacturers protocol. Arrays were scanned using the Agilent microarray scanner and raw signal intensities were extracted with their Feature Extraction v9.1 software. e data set was normalized using the quantile normalization method (2). No background subtraction was performed, and the median feature pixel intensity was used as the raw signal before normalization. e moderated t-statistic and false discovery rate for each gene of comparison between blood and spleen were calculated. Adjusted p-values were produced according to the Holm-Bonferoni method (3). All procedures were carried out using functions in the LIMMA software package of the Bioconductor Project (www.bioconductor.org). MIAME compliant expression data have been deposited under the accession GSE14850.

Real-time PCR reactions of preselected genes. For phenotypic differentiation of monocyte subsets by expression analysis we designed a TaqMan custom low–density array (Applied Biosystems) comprising 45 genes of interest and three endogenous control genes for quality controls purposes (Table S3). e technical details of the procedure can be found here: http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_040595.pdf. RNA was extracted from FACS-sorted monocyte subsets using the RNAeasy mini Kit (Qiagen). Typically, 250,000 monocytes of each subset were obtained from pooled blood and spleen samples of 10-15 mice. 250,000 cells yielded 100-250

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ng RNA in a volume of 35 µl. RNA yield and integrity were assessed with a NanoDrop spectralphotometer (ermo-Scientific) and an Agilent Bioanalyzer (Agilent) using the eukaryotic RNApico lab chip. Only samples with a RNA integrity number of above 7 were used for further processing. For low density array profiling, real time PCR cDNA was generated from 50 ng or 100 ng of RNA per sample by reverse transcription (RT) using multiplex RT pools (Applied Biosystems) according to the manufacturer’s protocol. e cDNA was then applied to the micro-fluidic card and real time PCR was performed on a 7900HT real time PCR machine (Applied Biosystems). Cycle threshold (Ct) values (auto thresholding) of the real time PCR readouts were compared among subsets derived from blood and spleen. Four independent replicates of each subset and from each organ were used for analysis. We used the Global Pattern Recognition (GPR) and geNorm algorithms to compare gene expression between groups. GPR detects significant changes in gene expression by multiple gene normalization, which does not require or assume constant level of expression of a single normalizer gene (i.e., ‘housekeeping gene’). By comparing the expression of each gene to every other gene in the array, a global pattern is established, and significant changes are identified and ranked (4). e geNorm algorithm implemented in the GPR program was used to calculate fold changes of gene expression based on the geometrical mean of a group of 10 best normalizers identified by GPR. Taken together, gene expression could be compared by two means: (i) ranking scores determining the significance of a difference in gene expression among the groups compared and (ii) by a reliable determination method of fold-changes in gene expression. Based on these readouts a gene was considered to be differentially expressed when it had been scored by GPR and showed a fold change >2.

In vitro phagocytosis. FACS-sorted monocytes from blood and spleen were incubated for 4 h with latex beads at a 1/10 cell/bead ratio (yellow-green latex beads, 2.5 µm, Sigma) in 200µl RPMI 1640 (Mediatech, Inc, VA) in a 96-well plate (100K/well) (Costar, Corning Inc, NY).

In vitro differentiation. FACS-sorted Ly-6Chigh and Ly-6Clow monocytes from blood and spleen were treated either with M-CSF (0.02 µg/ml) or GM-CSF (0.5 µg/ml) and IL-4 (0.2 µg/ml) in RPMI 1640 medium (Cellgro, Mediatech, Inc, VA) supplemented with 10% FCS (Valley Biomedical, Inc.), 50 µM 2-Mercaptoethanol (Cellgro, Mediatech, Inc, VA) and 100 U/ml Penicillin-Streptomycin (Cellgro, Mediatech, Inc, VA). Cells (105) were plated in triplicate in 96-well round-bottom plates (Costar, Corning Inc, NY) and cultured in a humidified incubator at 37°C, 5% CO2. e medium was replaced every second day to keep the growth factors fresh. Cells were harvested at day 7, and expression of the cell surface markers F4/80 and CD11c was determined by staining for 30 min with anti-F4/80-biotin, C1:A3-1 (AbD Serotec) and anti-CD11c-APC, HL3 (BD Biosciences); Strep-PerCP (BD Biosciences) labeled the biotinylated antibody.

Histology. Histology of spleens was assessed for the following groups: wild type C57BL/6 mice, wild type mice 1 day after MI, wild type mice 1 day after Ang II, Cx3cr1gfp/+ mice, Cx3cr1gfp/+ mice 1 day after MI, and Cx3cr1gfp/+ mice 1 day after Ang II. Spleens were excised, rinsed in PBS and embedded in OCT (Sakura Finetek). Fresh-frozen serial 6 µm thick sections were used for overall histological analysis and immunofluorescence staining. Hematoxylin and eosin staining was used to identify red and white pulps. Sections were incubated with anti-CD11b, M1/70 (BD Pharmingen); anti-F4/80, A3-1 (Abcam); anti-CD11c, 3.9 (Abcam); anti-neutrophil, NIMP-R14 (Santa Cruz Biotechnology, Inc); anti-CD163, G-17 (Santa Cruz Biotechnology, Inc); or anti-CD49b, Hal/29 (BD Pharmingen) antibodies followed by an appropriate biotinylated secondary antibody, and texas red-conjugated streptavidin (GE Healthcare). DAPI (Vector Laboratories) was used to identify cell nuclei. Negative controls were obtained by incubating tissue sections with the corresponding secondary antibodies only. Cell numbers were quantified using IPLab (version 3.9.3; Scanalytics, Inc., Fairfax, VA) and signal intensities were calculated using ImageJ (version 1.38x).

Parabiosis. Surgical gloves and autoclaved sterilized instruments were used. Animals were kept warm with a heating pad. Mice were weight-matched. We administered analgesia (buprenorphine 0.05-0.2 mg/kg) 30 minutes before surgery. Mice were anesthetized with isoflurane (2%/2L) and joined by a technique adapted from Bunster and Meyer (5). After shaving the corresponding lateral aspects of each mouse, matching skin incisions were made from behind the ear to the tail of each mouse, and the subcutaneous fascia was bluntly dissected to create about ½ cm of free skin. e olecranon and knee joints were attached by a mono-nylon 5.0 (Ethicon, Albuquerque, NM), and the dorsal and ventral skins were approximated by continuous suture. In some experiments, after an interval of several weeks, parabiosed mice were surgically separated by a reversal of the procedure. Percent chimerism was defined for gated monocytes as %CD45.1 / (%CD45.1 + %CD45.2) in CD45.2 mice, and as %CD45.2 / (%CD45.2 + %CD45.1) in CD45.1 mice.

Myocardial infarction. Mice or rats were anesthetized with gas anesthesia (isoflurane 2% / 2L O2), and intubated and ventilated with an Inspira Advanced Safety Single Animal Pressure/Volume Controlled Ventilator (Harvard Apparatus, Holliston, MA). e chest wall was shaved and left thoracotomy was performed in the 4th left intercostal space. e left ventricle was visualized and the left coronary artery was permanently ligated with monofilament nylon 8-0 sutures (Ethicon, Somerville, NJ) at the site of its emergence from under the left atrium. e chest wall was closed with 7-0 nylon sutures and the skin was sealed with superglue. Notably, for mice used in this study, the infarcts were of small to moderate size (~15% in delayed enhancement MRI) and therefore did not alter blood pressure or cardiac output. We measured cardiac index in infarcted and non-infarcted mice using gated high field cardiac MRI volumetry as described previously (6). Cardiac index was not changed on day 1 after coronary ligation: MI 780 ± 53 ml/min*kg, no MI 792 ± 94 ml/min*kg, n=6 per group.

Splenectomy. During isoflurane anesthesia, the abdominal cavity of mice was opened and the spleen vessels were cauterized. e spleen was carefully removed and placed in cold PBS solution. For control experiments, the abdomen was opened, but the spleen was not removed. In rats, splenic vessels were ligated with 7-0 sutures.

Spleen transplant. A pictorial representation of the procedure is shown in Figure S7. Spleen donor mice (CD45.2) were anesthetized with a subcutaneous injection of ketamine (90 mg/kg) and xylazine (10 mg/kg), followed by an intravenous injection of 200 units of heparin (American Pharmaceutical, Schaumburg, Il). e complete inhibition of clotting ensures that no vascular or intrasplenic thrombosis occurs. In deep anesthesia, the thorax was then opened and the right atrium incised to allow blood to exit during perfusion. Over a period of 5 minutes, the entire mouse was then perfused with a total of 20 ml of normal saline through a cannula inserted into the apex of the left ventricle. At the end of this procedure, fluid exiting the right atrium was clear which indicated thorough removal of the donor blood. e abdomen of the donor was then opened with a longitudinal incision. e pancreas, the spleen and the abdominal vasculature in the epigastric region were visualized. Small vessels between the pancreas and the intestine were ligated with 6.0 cotton (Ethicon). e celiac artery was then isolated, and the hepatic and gastric artery ligated with 10.0 suture (Ethicon). e abdominal aorta was ligated and cut just below the celiac artery with micro-dissection scissors (ROBOZ, Rockville, MD), and dissected above the celiac artery. is approach resulted in an aortic cuff connected to the splenic artery, which allowed vascular anastomosis of the spleen to the recipient. Following ligation of the bile duct, the portal vein was isolated, and the superior and inferior mesenteric and gastric veins were ligated. e portal vein was intersected closely to the liver. e entire organ package containing the vascular connections, spleen and the pancreas was then removed and stored in ice cold saline for 15 minutes while the recipient was prepared. e recipient (CD45.1) was anesthetized with isoflurane supplemented with oxygen (2-3 Vol%). An abdominal midline incision was made and the inferior vena cava and the descending aorta were isolated below the

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renal arteries. e recipient vessels were clamped with an atraumatic bulldog clamp (ASSI, Westbury, NY) and opened with micro-scissors. e portal vein was anastomosed to the inferior vena cava and the donor aortic cuff was connected with an end-to side anastomosis to the recipient aorta using 10.0 suture. e clamp was then removed to restore blood flow. e time of ischemia of the donor spleen, which ended after completion of both vascular anastomoses by unclamping the recipient aorta and vein, was ~60 min. Flow cytometric analysis of transplanted spleens (in mice without MI) indicated that the procedure reduced on average the reservoir of donor splenic monocytes by ~6-fold (donor monocytes in donor spleens 12 h after operation: 0.23 ± 0.01 X 106 cells, n=2; control monocytes in control spleens: 1.4 ± 0.2 X 106 cells, n=8). e ‘missing’ monocytes in transplanted spleens likely matured into Mø/DC because the number of these cells increased locally (donor CD45.2+ CD11b+ Mø/DC in donor spleens: 0.99 ± 0.6 X 106 cells; control CD11b+ Mø/DC in control spleens: 0.36 ± 0.5 X 106 cells). Some monocytes may also have died locally, however they virtually did not enter circulation (donor monocytes in blood: 0.0012 ± 0.0004 X 106

cells). e reduced availability of donor monocytes in transplantation experiments was taken into account when quantifying their relative contribution in infarcts (see below). In an additional cohort of mice, 1 h after this procedure, the mouse was re-anesthetized and myocardial infarction was induced as described above (n=2). 24 h later, flow cytometry analysis of cells from MI revealed 10.1 ± 2% monocytes of splenic origin. Taking into account the reduced reservoir of splenic monocytes in transplanted animals, we calculated that a normal spleen should contribute ~40% of the recruited monocytes (6 X 10.1% splenic monocytes versus 89.9% other monocytes ≈40% splenic monocytes versus 60% other monocytes). We also performed the two following control experiments: e first experiment involved mice (n=2) transplanted with a spleen as mentioned above, but the spleens were excised just before unclamping the host vasculature, leaving only the donor vasculature, connective tissue and parts of the pancreas in the recipient mouse. e mice were then subjected to MI and the infarcts investigated 24 h later by flow cytometry. We detected no CD45.2+ cells in these mice. e second experiment involved mice (n=2) that received a spleen graft but were not subjected to MI. In some experiments (fig. S11), donor spleens were transplanted into the great omentum of recipients. Specifically, awhile submerged in ice-cold PBS solution, donor spleens were sectioned into 5 pieces of approximately 3 mm thickness, transplanted, and fixed in place with 8-0 nylon sutures. To facilitate microvascular connections, the hilar aspect of the splenic segments was brought in proximity to the great omental vessels. e institutional Subcommittee on Research Animal Care at Massachusetts General Hospital (MGH) approved all animal studies.

Fluorescence Molecular Tomography (FMT) - Magnetic Resonance Imaging (MRI). Animal preparation: Mice were shaved and depilated to remove all hair that otherwise absorb light and interfere with optical imaging. Mice were anesthetized (Isoflurane 1.5%, O2 2 L/min) and rendered immobile in a multimodal imaging cartridge, which lightly compresses the anesthetized mouse between optically translucent windows. e latter do not interfere with fluorescence imaging. e cartridge provides fiducial landmarks on the frame that enable exact, robust and observer-independent fusion of images. e cartridge also prevents motion of the mouse during transfer between modalities. CLIO-680 and ProSense-750 (VisEn Medical) were injected intravenously on day 1 after coronary ligation at a dose of 15 mg iron oxide/kg body weight and 5 nmol respectively, and FMT-MRI was carried out 24 hours later. Twelve mice with MI were imaged; 6 received splenectomy at the time of coronary ligation. A commercially available FMT system was used for in vivo imaging studies (FMT2500, VisEn Medical). e system allows for dual-channel imaging without the need for an index matching fluid, i.e., is a noninvasive free-space imaging system. It is equipped with MRI-safe mouse holders, allowing for imaging in the FMT and MRI systems without the need to reposition the mouse. Typical FMT scan times were less than 5 min per mouse. Data were post-processed using a normalized Born forward equation to

calculate a 3D map of fluorochrome concentration (7). To reliably identify the region of interest within the heart, FMT was hybridized with MRI for anatomic reference. After completion of FMT, the imaging cartridge holding the anesthetized mouse was inserted into a custom-made plexiglass holder that supplies isoflurane anesthesia and optimal positioning inside a transmit-receive MR body coil. We used a 7 Tesla horizontal bore scanner (Pharmascan, Bruker, Billerica) and a RARE sequence (TE 26.9 ms, TR 2500 ms, slice thickness 1 mm, 24 slices, matrix 256x256, FOV 6x6 cm) to image the entire mouse and the fiducial markers on the imaging cartridge. e fiducial wells were filled with fluorochrome solution, and therefore were readily identified by FMT (fluorescence) and MRI (proton signal). FMT and MRI DICOM images were fused with OsiriX (e Osirix Foundation, Geneva, Switzerland). Within both data sets, fiducial points were tagged to define their X-Y-Z-coordinates. Using these coordinates, FMT data were then resampled, rotated and translated to match the MR image matrix, and finally fused. After identification of infarcts on MRI, regions of interest were defined in both FMT channels. FMT-derived fluorescence is given in pmol and represents the absolute quantity of the excited fluorochrome within the infarct.

Fluorescence reflectance imaging (FRI). Myocardial short-axis sections were produced after harvest of rat hearts and then exposed on a custom-built fluorescence reflectance system 24 h after i.v. injection of 5 mg/kg CLIO-VT680.

Delivery of Angiotensin (Ang) II. C57BL/6 mice were implanted subcutaneously in the dorsum of the neck with osmotic mini-pumps (Alzet) for 2 to 24 hours. Mini-pumps were pre-incubated in PBS for 4 h to assure immediate delivery of the agent after implantation. Ang II (Bachem) was delivered at a rate of 1 µl/h at 1.5 mg/kg/day. Control mice were implanted with mini-pumps delivering saline (0.9% NaCl) (n=3-6).

ELISA for determination of serum Ang II levels. Blood was obtained from mice under anesthesia by cardiac puncture with a syringe pre-loaded with 80 µl of 100 mM EDTA anticoagulant. e blood was supplemented with 50mM p-hydroxymercuribenzoid acid (Sigma), centrifuged, and supernatant loaded onto Amprep Phenyl PH mini-columns (GE Biosciences) to isolate peptides from the sera. Methanol-eluted peptides were dried by vacuum centrifugation. Ang II concentration was determined by using an Ang II ELISA (Cayman Chemical), and normalized to the volume of blood isolated.

Western for Ang II Type 1 (AT-1) receptor on splenic monocytes. Monocytes were isolated by FACS as described above. Sample pellets of ~175,000 monocytes were resuspended in Laemmli buffer (BioRad), and sonicated to lyse cells and shear genomic DNA. Samples were developed by electrophoresis on a 4-15% polyacrilamide gel (BioRad). e proteins were transferred to a polyvinylidene difluoride membrane (Fischer) by semidry transfer. Membranes were blocked with carnation milk and PBS supplemented with 0.05% Tween 20 overnight. Membranes were washed, stained initially with anti-AT-1 receptor antibody (Abcam), stripped with Restore buffer (Pierce), and stained with anti-glyceraldehyde-3-phosphate (GAPDH) (Rockland Immunochemicals for Research). Blots were developed with Western Lightning Chemiluminescence reagent (PerkinElmer Instruments) and molecular weights were compared to bands for Precision Plus Protein Western C standards (BioRad).

In vitro migration. Migration experiments using Ang II as a chemoattractant were performed in BD BioCoat invasion chambers (BD Biosciences). Sorted monocytes from spleen were suspended in RPMI 1640 media (Cellgro) supplemented with 0.2% FCS (Valley Biomedical, Inc.). 2x105 cells were placed on the matrigel-coated 8 µm pore size PET membrane and incubated in a humidified incubator at 37°C, 5% CO2 for 1 h, allowing the cells to attach to the matrigel. Migration was induced by addition of Angiotensin II (1 µM) (Bachem) to the lower compartment. After 2 h, non-migrating cells were removed with a cotton

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tip and the membranes were fixed and stained with HEMA 3 staining set (Fisher Scientific) to identify cells that had migrated to the lower surface of the membrane. e number of migrated cells was determined per 200 × high-power field. Cells that had migrated to the lower chamber were counted using Trypan Blue (Cellgro).

Intravital imaging. Animal preparation: During isoflurane anesthesia, the peritoneal cavity was opened with a transverse incision in the disinfected abdominal wall. e gastric-splenic ligament was dissected and the spleen carefully exteriorized. Robust blood flow was observed in the splenic artery during the duration of each experiment and splenic perfusion was confirmed by inspection through fluorescence microscopy upon tail vein injection of an intravascular imaging agent. e exteriorized spleen was completely submerged in temperature-controlled saline solution. Temperature near the spleen was carefully monitored using an Omega HH12A thermometer with fine wire thermocouples (Omega Engineering Inc., Stamford, CT) and kept at 37ºC. Confocal Microscopy: Images were collected with a prototypical intravital laser scanning microscope (IV100, Olympus Corporation, Tokyo, Japan) (8) using an Olympus 20x UplanFL (NA. 0.5) objective and the Olympus FluoView FV300 version 4.3 program. Samples were excited at 488 nm with an air-cooled argon laser (Melles Griot, Carlsbad, CA) for visualization of the GFP+ cells, and at 748 nm with a red diode laser (Model FV10-LD748, Olympus Corporation, Tokyo, Japan) for visualization of the blood pool agent (AngioSense-680, VisEn Medical, MA). Light was collected using custom-built dichroic mirrors SDM-570 and SDM-750, and emission filters BA 505-550 and BA 770 nm IF (Olympus Corporation, Tokyo, Japan). Both channels were collected simultaneously. A prototypical tissue stabilizer (Olympus Corporation, Tokyo, Japan) was used to reduce motion and stabilize the focal plane. e stabilizer was attached to the objective and its z-position was fine adjusted using a micrometer screw to apply soft pressure on the tissue. Time-lapse recordings were made by collecting images of 256x256 pixels at 15 s intervals over 1 h in a single plane (2D) of focus. Mice were analyzed in the steady-state or 2 h or 24 h

after either MI or infusion of Ang II. Multiphoton Microscopy: Mice were anesthetized with ketamine (150 mg/kg BW) and xylazine (10 mg/kg BW) i.m., and the spleen was immobilized by placing a coverslip on its ventral surface. Images were collected with Praireview software on an Ultima IV upright multiphoton microscope (Prairie Technologies, Middleton, WI) equipped with an Olympus 20x/0.95 NA water immersion objective. For multiphoton excitation and second harmonic generation, a Ti:sapphire laser with 10-W MilleniaXs pump (Mai Tai HP, Spectra-Physics, Mountain View, CA) was tuned to 920 nm. Emitted light and second harmonic signals were detected through 525/50 and 460/50 nm bandpass filters using non-descanned detectors to generate two-color images and stacks, which were volume-rendered using the brightest-spot rendering mode within Volocity software (Improvision, Coventry, UK). Optical slides were acquired at 1 or 2 µm intervals. e number of stacks varied between 16 and 60 (please refer to Movies S1-3 for more information). Data Analysis: All GFP+ cells were identified manually in each recording. To determine the displacement over time of individual cells, the centroid position (x-y dimension) of these cells was recorded at the first and last time-point when they could be identified during a recording; then the distance between these two points was calculated, and divided by the elapsed time. Single cell tracks for GFP+ cells were generated based on the position of cell centroids from a series of images recorded at 15 s intervals, and ImageJ and the Manual Tracking plugin (http://rsbweb.nih.gov/ij/plugins/track/track.html) were used for display and quantification. Motion-artifacts in recordings were corrected using the auto-alignment plugin (stackreg) of ImageJ (http://rsb.info.nih.gov/ij/).

Statistics. Results were expressed as mean±SEM. Statistical tests included unpaired, 2-tailed Student's t test using Welch's correction for unequal variances and 1-way ANOVA followed by Bonferroni’s multiple comparison test. P values of 0.05 or less were considered to denote significance.

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Supplementary Figures

Figure S1: Co-existence of macrophages/DCs and undifferentiated monocytes in the spleen. Leukocytes were retrieved from the spleen and labeled with antibodies as described in Methods. (A) Gating on leukocytes based on CD11b, Lin (i.e. CD90/B220/CD49b/NK1./Ly-6Glow), F4/80 and CD11c expression identifies at least eight different populations, two of which (CD11b+ Linlow F4/80low CD11c– Ly-6Chigh/

low) are monocytes. (B) Enumeration of the eight populations reveals different numbers of monocytes, macrophages (Mø; defined by F4/80 expression), DCs (DC; defined by CD11c expression) or other cells (many of which are lymphocytes) (n=3). (C) Enumeration of all monocytes vs all macrophages or DCs in the spleen (n=3). (D) CD11bhigh Linlow monocytes correspond to CD11bhigh CD115+ monocytes. e dot plot overlay shows that 97.5% of gated CD11bhigh CD115+ cells (blue dots) overlay with CD11bhigh Linlow monocytes. All data are from one experiment representative of at least three independent experiments.

Figure S2: Flow cytometry identification of various immune cell types obtained from the spleen of Cx3cr1gfp/+ mice. Monocytes were defined as CD11bhigh Linlow (F4/80/CD11c/I-Ab)low and were further divided into Ly-6Chigh/low subsets. DC were defined as CD11c+ cells; Mø as F4/80+; NK cells as (NK1.1/CD49b)high; T cells as CD3+; B cells as B220high; and neutrophils as SSChigh Ly-6Ghigh. EGFP expression was analyzed for each cell type; mean fluorescence intensities (MFI) are shown on the right (n=3). Of note, 6±3% NK cells were EGFP+, whereas virtually all monocytes were EGFP+. When taking into account the abundance of each cell population and the fractions that are EGFP+, our flow cytometry data indicate that EGFP+ monocytes outnumber EGFP+ NK cells by >12 to 1 in the spleen (i.e., 92% EGFP+ monocytes for 8% EGFP+ NK cells). All data are from one experiment representative of at least two independent experiments.

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Figure S3: Monocytes reside in the subcapsular red pulp. (A) Immunofluorescence panels of spleens from Cx3cr1gfp/+ mice flash-frozen and stained with anti-CD11b, F4/80, CD11c, CD163 or CD49b (red) antibodies and DAPI (blue), and co-localized with GFP (green). e subcapsular red pulp (left three panels) and marginal zones (right three panels) are shown; srp = subcapsular red pulp, mz = marginal zone, wp = white pulp. We used the same exposure times to capture fluorescence of a given Ab in SRP and MZ. Monocytes were GFPhigh, CD11b+, F4/80low, CD11c– and CD163– and were found predominantly in the subcapsular red pulp. GFP– CD11bhigh population represents neutrophils. Very few CD49b+ GFP+ NK cells were observed in the subcaspular red pulp. CD163 identifies iron-recycling macrophages that reside predominantly in the SRP. (B) Negative controls were generated by staining with the appropriate secondary antibodies only. (C) Signal intensity of GFP, CD11b, F4/80 and CD11c in various regions of the spleen. e numbers were generated by drawing same-area regions of interest with ImageJ on

regions either in the subcapsular red pulp or the marginal zone that were positive for each signal, so as to calculate intensity of a given signal as a measure of expression for that particular marker (note that exposure times were kept the same for any given marker in all regions). e data show that GFP+ cells are most bright in the subcapsular red pulp, in keeping with flow cytometry data in Fig 2A that show that monocytes are brightest compared to macrophages. Also note that F4/80 and CD11c expression is brightest in the marginal zone, in keeping with flow cytometry data of Fig S2, which show that mature macrophages express F4/80 higher than monocytes (notably, monocytes are F4/80–/low) and mature DCs express CD11c higher than monocytes (monocytes are predominantly CD11c–; see Figure 1H). CD11b and CD163 expression are similarly bright in both regions. *P<0.05; **P<0.001 (n=10 high power fields). All data are from one experiment representative of at least two independent experiments.

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Figure S4: Splenic monocytes reside in the spleen and thus do not traffic through the spleen while in the blood stream. (A) Intravital microscopy investigation of splenic monocytes in mice devoid of blood leukocytes. (1) Cx3cr1gfp/+ mice received an intravenous injection of a fluorescent blood pool agent (AngioSense-680). (2) Intravital imaging of the spleen subcapsular red pulp shows blood vessels (red), as well as monocyte clusters (green) outside the vessels. (3) Blood from these mice was then removed by injecting 20 ml saline into the left ventricle. Blood was allowed to exit on the venous side through a small excision performed in the right atrium. e inserts on the lower right of the quadrant show pictures of venous samples that were obtained before and after the flushing procedure. Note that the samples obtained after the flushing procedure are clear and virtually devoid of leukocytes (flow cytometry analysis indicates that the few remaining cells are all red blood cells). us, the perfused mice did not contain circulating blood monocytes. (4) e same mice were subjected again to imaging. As expected, the extensive flushing of the vessels with saline removed the blood pool agent in the spleen vessels. Importantly, however, the subcapsular red pulp retained splenic monocytes. e topography of these cells is indistinguishable from the one observed before perfusion. (B) Flow cytometry analysis of blood samples and spleen in mice that were flushed with 20 ml saline or not. Blood data show the number of monocytes per ml of blood (before flush), or per ml of saline (after flush). Spleen data show the total number of monocytes in spleens of animals that were flushed or not (n=3-5).

Figure S5: Monocyte exchange in parabiotic mice during parabiosis and after surgical separation. e cartoons illustrate different groups of mice that were subjected to parabiosis. Parabiosis was established between mice expressing two distinct CD45 allotypes (CD45.1 and CD45.2). Mice were divided into 3 groups (4 mice per group). e first and second groups were sacrificed 2 and ~30 days post joining. Blood mixing initiates at day ~2 and thus is useful to measure monocyte exchange between parabionts. e percent chimerism of monocytes has reached a plateau by day ~30 (9). e third group of mice involved separation of parabionts at day ~30 and sacrifice 18h later. Separation of mice prevents further exchange between parabionts and thus is useful to measure the rate of monocyte turnover and replacement. e graphs at bottom depict the percent chimerism of monocytes in blood and spleen of each parabiont. e fold difference in percent chimerism between blood and spleen is shown on top of each graph (e.g., 13:1 means that the percent chimerism is 13-times higher in blood than in spleen). e experiment reveals that, early after joining, the percent chimerism of monocytes was lower in spleen than in blood, indicating that monocytes actively seeded the splenic niche. Monocytes also resided longer in spleen than in blood, because the percent chimerism of monocytes was higher in spleen than in blood in long-term parabionts that had been separated recently. All data pool at least two independent experiments.

Figure S6: e decreased number of splenic monocytes after MI is not linked with increased cell death or with an increased number of splenic Mø/DC. (A) Total number of splenic monocytes in resting mice (‘no MI’), or 1 day post MI. (B) Percentage of splenic monocytes that are Annexin-V+ or Propodium Iodide+. (C) Total number of splenic Mø/DC. e results suggest that after MI, splenic monocytes exit the spleen rather than die or mature into Mø/DC locally (n=3). **P < 0.005.

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Figure S7: Splenectomized animals show normal monocyte and lymphocyte counts in blood and bone marrow. (A) Number of blood monocytes per ml of blood (left) or of bone marrow monocytes per tibia (right) in control mice (‘+Spleen’) or in splenectomized animals (‘–Spleen’). (B) Number of blood and bone marrow lymphocytes in the same groups of mice. Mice were analyzed by flow cytometry. Splenectomized mice were sacrificed 1 day after surgery (n=3).

Figure S8: Analysis of monocytes in rats subjected to MI. (A) Flow cytometry analysis of spleens from control rats and from rats subjected to MI. (B) Total count of CD11b+ cells in spleen as identified in panel A. (C) Flow cytometry analysis of enzymatically-digested hearts 1 day after MI in rats with or without spleen. (D) Total count of CD11b+ cells in hearts as in panel C. n=3–4. (E) Fluorescence reflectance imaging (FRI) of explanted hearts 1 day after MI obtained from rats with or without spleen. Rats received an intravenous injection of CLIO-VT680 at the time of MI (e.g., 1 day before sacrifice). FRI at 680 nm wavelength measures fluorescent nanopartice uptake; FRI at 488 nm wavelength measures autofluorescence. (F) Target to background ratio in the infarcted hearts shown in panel E (n=3–4). *P < 0.05; ***P < 0.0005.

Figure S9: Spleen transplantation with vascular anastomosis. A spleen transplant model was developed to quantitatively track splenic monocytes after induction of myocardial infarction. (1) e spleen was harvested from a CD45.2+ mouse after inhibition of clotting with heparin. Before explantation, the donor animal was perfused with saline until no blood remained in circulation. (2) e donor spleen was then carefully exposed using micro-dissection tools and a surgical microscope. To facilitate vascular anastomosis to the recipients’ circulation, the splenic artery and vein were prepared while they remained connected to the abdominal aorta and the portal vein. e use of this vasculature with a much higher caliber as a conduit facilitated vascular anastomosis of the splenic artery and vein to the recipient. Side branches and other major vessels were carefully ligated to avoid bleeding. (3) e organ was then transplanted into the intraperitoneal cavity of the CD45.1+ recipient, and arterial and venous anastomoses were produced using microsurgical techniques. End-to-side anastomoses of the aortic cuff to the recipient descending aorta, and of the portal vein to the recipient inferior vena cava were established while these vessels were clamped. After unclamping, flow through the transplanted organ was verified visually, followed by splenectomy of the orthotopic CD45.1 recipient spleen. Vascular anastomosis of splenic vessels is shown on the digital image of the surgical field on the right. To better visualize the anatomy, the spleen was flipped over to the right side of the animal. (4) e abdomen was then closed and myocardial infarction was induced in the recipient by coronary ligation through a left lateral intercostal thoracotomy. (5) 12 hours later, the infarct was harvested and prepared by enzymatic digestion. (6) e resulting cell suspension was stained with an antibody cocktail including antibodies for CD45.1 and 2, which reported the source of recruited monocytes by flow cytometry. CD45.2+ cells originated from the donor spleen, whereas CD45.1+ cells were from the recipient animal.

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Figure S10: Number of monocytes originating from a transplanted donor spleen and released into blood of the recipient. e data show the number of total monocytes in 2 ml blood. Mice were analyzed in transplanted animals that were subjected to MI or not (n=2).

Figure S11: e spleen is not a site of monocyte production. CD45.2+ donor spleens were implanted in the omentum of CD45.1+ mice and removed for analysis 1 day or 21 days after transplantation. Data show representative contour plots depicting the relative distribution of donor CD45.2+ and host CD45.1+ cells (n=3). *P < 0.05.

Figure S12: Splenic monocytes and neutrophils have distinct reservoir capacities. (A) Flow cytometric enumeration of neutrophils in single cell suspensions obtained from the entire spleen, and either in the absence of MI or 0.5 and 1 d after MI. e number of neutrophils is statistically unchanged after MI, which indicates that the spleen does not lose a significant population of neutrophils (n=3-6). (B, C) Immunofluorescence analysis of neutrophils (NIMP-R14+ cells) in the subcapsular red pulp in the absence of MI or 1 d after MI. Panel B shows a similar pattern of neutrophil residency, and Panel C shows similar neutrophil counts 1 day after MI when compared to controls. (D) Flow cytometric analysis of neutrophil counts in the blood in the absence of MI or 0.5 and 1 day after MI in mice with or without spleen. e results show no significant contribution of the spleen to the number of circulating neutrophils (n=6). (E) Flow cytometric analysis of neutrophils in the heart either in the absence of MI or 1 day after MI in mice with or without spleen. (F) Enumeration of neutrophils in the same mice shows a

slight, but insignificant decrease of neutrophils in mice without spleen. n=3-6. (G) CD45.2+ spleens were transplanted immediately after MI to CD45.1+ splenectomized mice. Data show that 1 day after MI spleen-derived neutrophils do not contribute to the neutrophil population in the heart. Data pool two experiments (A, D, F), or are from one experiment representative of two independent experiments (B, C, E, G).

Figure S13: Redistribution of monocyte subsets after MI. Total number of Ly-6Chigh and Ly-6Clow monocytes lost from the spleen (left) and gained in the heart (right) 1 day after MI. Mean±SEM (n=6–15). Data pool at least three independent experiments.

Figure S14: Accumulation of monocytes in heart of Atgr1a–/– mice. Control (WT) and Atgr1a–/– mice were subjected to MI. e number of monocytes per mg tissue was determined 1 day later (n=3). *P<0.05.

Figure S15: CD11b+ cells emigrate the subcapsular red pulp in response to Ang II. (A) Representative immunofluorescence sections of the spleen stained with anti-CD11b (red) and DAPI (blue) depict the subcapsular red pulp from control mice (left) and mice 1 day after Ang II osmotic pump infusion (right). (B) Enumeration of CD11b+ cells per high power field in the subcapsular red pulp of the mice mentioned above. Mean and SEM are shown, n=10 high power fields. (C) Intravital microscopy of GFP+ cells (green) in the spleen subcapsular red pulp of live Cx3cr1gfp/+ mice. Images show clusters of monocytes in control mice (left) and their disappearance 1 day after Ang II osmotic pump infusion (right). *P<0.05. All data are representative of at least two independent experiments.

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Figure S16: Identification of GFP+ Mø/DC, patrolling monocytes, and splenic monocytes by intravital microscopy in Cx3cr1gfp/+ mice. (A) Representative immunohistology of a large F4/80high GFP+ Mø/DC (top) and a smaller F4/80lo/– GFP+ monocyte (bottom). (B) GFP+ cells recorded in the subcasular red pulp were divided based on their size (Mø/DC: Area ≥150 µm2, ~5% of recorded GFP+ cells; monocytes: Area <150 µm2, ~5% of GFP+ cells). (C) After using size to separate GFP+ cells into Mø/DC and monocytes, monocytes were further divided into patrolling monocytes (cells physically interacting with blood vessels, ~5% of GFP+ cells), and splenic monocytes (not physically interacting with vessels, ~90% of GFP+ cells). It is worth noting that splenic monocytes represent the vast majority of recorded GFP+ cells. Mø/DC were in minority because our recordings were performed in the subcapsular red pulp (marginal zones are typically located at depths greater than 100 µm). (D) Mø/DC show on average slower mean track velocities than splenic monocytes (Mø/DC: 0.20 ± 0.10 µm/min; splenic monocytes: 2.28 ± 2.95 µm/min; t test: P<0.028). (E) Mø/DC show on average a lower circularity (or ‘roundness’) index than splenic monocytes (Mø/DC: 0.48 ± 0.12; splenic monocytes: 0.78 ± 0.09; t test: P<0.0001). Circularity Index = 4πA/P2, where A is the cell cross-sectional area and P the cell perimeter). Data pool (B, D, E) or are from one experiment representative of three independent experiments (A, C).

Figure S17: Displacement over time for all parenchymal GFP+ cells in Cx3cr1gfp/+ mice. Data show displacement over time for all splenic GFP+ cells, i.e., both splenic monocytes and splenic Mø/DC, in resting mice (control) and 1 day after MI or infusion of Ang II. n=155 (control), 171 (MI), and 134 (Ang II) cells. is figure illustrates that the combination of so-called ‘splenic monocytes’ and ‘splenic macrophages/DCs’ (analyzed separately in Fig 4I) preserves statistically different displacements over time between control and MI-treated or Ang II-treated groups. *P<0.01, **P<0.001. Data pool three independent experiments.

Figure S18: Activation of splenic monocytes in vivo occurs without physical contraction of the subcasular red pulp. (A) Anesthetized Cx3cr1gfp/+ mice received a blood pool agent to visualize vessels (Angiosense-680, red) and were imaged by intravital micrcoscopy at relatively low magnification in monocyte-rich regions of the subcapsular red pulp. (B) Mice then received Ang II as described previously and the same region was imaged at different time points. Images on top show intravital recordings, whereas images on the bottom include the outline of vessels identified at time=0. e outline serves as a fiducial marker and is reproduced on the subsequent images. Physical contraction of the subcapsular red pulp should associate with decreased distances between the fiducial marker, a phenomenon that we did not observe during the duration of the experiment. Note leakage of the blood pool agent in the red pulp parenchyma over time, which is in line with previous findings that Ang II increases microvascular permeability.

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Supplementary Tables

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Legends to Supplementary Movies Movie S1: Ring of GFP+ macrophages/DCs in the marginal zone of the spleen. e animation shows GFP+ macrophages/DCs (green) at different depths throughout the spleen from Cx3cr1gfp/+ mice, starting at 50 µm and ending at 110 µm below the capsule. e cells are arranged in a ring in the marginal zone surrounding the white pulp. Scale bar=50 µm.

Movie S2: GFP+ monocytes in the subcapsular red pulp of the spleen. e animation shows GFP+ monocytes (green) in the subcapsular red pulp of the spleen from Cx3cr1gfp/+ mice. e dense collagen network (blue) of the splenic capsule indicates the boundary of the organ. e recording covers 120 µm at 2 µm increments. Scale bar=100 µm.

Movie S3: GFP+ monocytes in the subcapsular red pulp of the spleen. e animation shows GFP+ monocytes (green) in the subcapsular red pulp of the spleen from Cx3cr1gfp/+ mice, starting at 20 and ending at 50 µm below the capsule, respectively. Collagen fibers are shown in blue. Scale bar=25 µm.

Movie S4: A splenic monocyte in the subcapsular red pulp. e moviemovie shows a GFP+ splenic monocyte (green) in the subcapsular red pulp of a live Cx3cr1gfp/+ mouse. A fluorescent blood pool agent was injected immediately before imaging and is shown in red. Tracks indicate the position of the cell centroid at 15 second intervals. Time is shown in minutes and seconds. Scale bar=20 µm.

Movie S5: A splenic DC or macrophage in the subcapsular red pulp. e movie shows a GFP+ DC or macrophage (green) in the subcapsular red pulp of a live Cx3cr1gfp/+ mouse. A fluorescent blood pool agent was injected immediately before imaging and is shown in red. Tracks indicate the position of the cell centroid at 15 second intervals. Time is shown in minutes and seconds. Scale bar=20 µm.

Movie S6: A patrolling monocyte in the red pulp of the spleen. e movie shows a GFP+ monocyte (green) patrolling a blood vessel (red) in the subcapsular red pulp of a live Cx3cr1gfp/+ mouse. Blood vessels were visualized by injecting a fluorescent blood pool agent immediately before imaging. Tracks indicate the position of the cell centroid at 15 second intervals. Time is shown in minutes and seconds. Scale ba =20 µm.

Movie S7: Migratory activity of GFP+ cells in the subcapsular red pulp of the resting spleen. e movie on the left shows GFP+ cells (green) in the subcapsular red pulp of a live Cx3cr1gfp/+ mouse. A fluorescent blood pool agent was injected immediately before imaging and is shown in red. Tracks indicate the position of the centroid of all GFP+ cells at 15 second intervals. Movies on the right separately show tracks (top) and cells (bottom). ree types of GFP+ cells can be visualized: i) motile monocytes patrolling the blood vessel are located in the upper left region of the recording; ii) a sessile cell (labeled with a purple dot at the center of the recording), that is larger in size and has several cytoplasmic protrusions/dendrites, and thus likely represents a macrophage or DC; iii) monocytes located in the spleen parenchyma and not associated with blood vessels. Time is shown in minutes and seconds. Scale bar=20 µm.

Movie S8: Migratory activity of GFP+ cells in the subcapsular spleen red pulp 2 h after MI. e movie on the left shows GFP+ cells (green) in the subcapsular red pulp of a live Cx3cr1gfp/+ mouse. A fluorescent blood pool agent was injected immediately before imaging and is shown in red. Tracks indicate the position of the centroid of each GFP+ cell at 15 second intervals. Movies on the right separately show tracks (top) and cells (bottom). Time is shown in minutes and seconds. Scale bar=20 µm.

Movie S9: Migratory activity of GFP+ cells in the subcapsular spleen red pulp 2 h after infusion of Ang II. e movie on the left shows GFP+ cells (green) in the subcapsular red pulp of a live Cx3cr1gfp/+ mouse. A fluorescent blood pool agent was injected immediately before imaging and is shown in red. Tracks indicate the position of the centroid of each GFP+ cell at 15 second intervals. Movies on the right separately show tracks (top) and cells (bottom). Time is shown in minutes and seconds. Scale bar=20 µm.

Movie S10: Departing splenic monocyte. e movie shows a splenic GFP+ monocyte migrating from the spleen parenchyma to the lumen of a vessel. e cell likely had entered the vessel at time point 52:30 min:sec, and then migrated ~220 µm during the next 6 min. is represents an average instantaneous velocity of ~16 µm/min. is is in range with the average instantaneous velocities observed for patrolling monocytes. However, starting at time point 58:45 and during the next 30 sec, the cell migrated over >125 µm and disappeared from the field of view. is represents an average instantaneous velocity of >250 µm/min, which is one order of magnitude higher than the one reported for patrolling monocytes. Conversely, the cell did not enter free flow immediately after intravasation since it could be detected relatively close to the site of entry as late as 6 min after intravasation. us, departing monocytes may adhere for some time to the endothelium before they enter free flow. Tracks indicate the position of the cell centroid. Time is shown in minutes and seconds. Scale bar=20 µm.

Supplementary references1. S. Jung et al. Mol Cell Bio 20, 4106 (2000).2. B. M. Bolstad et al. Bioinformatics 19, 185 (2003).3. S. Holm, Scand J Stat 6, 65 (1979).4. S. Akilesh, et al. Genome Res 13, 1719 (2003).5. E. Bunster, R.K. Meyer, Anat Rec 57, 339 (1933).6. M. Nahrendorf et al. Circulation, 113:1196 (2006).7. M. Nahrendorf et al., Circ Res 100, 1218 (2007).8. H. Alencar, et al. Neoplasia 7, 977 (2005).9. K. Liu, et al. Nat Immunol 8, 578 (2007)

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