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1 Supporting Information Idoyaga et al /pnas SSC a) Single cell suspension 99 Aqua b) Live cells 96 -W c) Singlets 92 -A CD19+ER119 d) CD19 ER119 cells 97 CD3 e) CD3 cells 27 f) DX5 cells 71 DX5 g) high conventional DCs h) dim CD11b dim 14 j 16 F4/80 k PDCA h 18 j) F4/80 high red pulp macrophages k) PDCA-1 + B220 + plasmacytoid DCs i g CD11b 43 SSC CD11b m 36 i) dim CD11b high l 65 Ly6G m) Ly6G cells SSC n CD11b l) Ly6G + cells: granulocytes n) CD115 + cells: monocytes CD115 B220 Fig. S1. Gating strategy to identify cell subsets in normal mouse spleen. otal splenocytes from BALB/c mice were stained with different cocktails of antibodies to identify DCs (, CD11b, PDCA-1, B220, CD3, CD19, er119, DX5, CD8) or monocytes/macrophages (, CD11b, F4/80, Ly6G, CD115, CD3, CD19, er119, DX5). Among single cells (gate a), the live cells (gate b), singlets (gate c), and CD19, er119, CD3, and DX5 cells (gates d, e, and f) were selected for further analysis and examined for the expression of and CD11b. he high population (gate g) represents classical DCs (both CD11b high and CD11b low ). dim CD11b dim (gate h) were further analyzed for F4/80 expression to identify RPMs (gate j; F4/80 high, dim, CD11b dim ) [aylor et al. (2005) Annu Rev Immunol 23: ] and for the expression of PDCA-1 and B220, which results in the identification of plasmacytoid DCs (gate k; B220 PDCA-1, int, CD11b dim ) [Villadangos and Young (2008) Immunity 29: ]. CD11b high cells (gate i) were further examined for Ly6G expression, dividing them into 2 groups: Ly6G, which represents granulocytes (gate l) [Daley et al. (2008) J Leukocyte Biol 83:64 70] and Ly6G cells (gate m). Cells within the Ly6G fraction (gate m) were analyzed for the expression of CD115 identified as monocyes (gate n) [Randolph et al. (2008) Curr Opin Immunol 20:52 60]. 1of6

2 A YG Polystyrene MESH B YG Polystyrene MESH CD8 Fig. S2. DCs accumulate few fluorescent latex particles given intravenously. Sections of spleen were analyzed 30 min after the inoculation of YG-PS intravenously. Spleen sections were stained with - (A, red) or -CD8 (B, red) and Alexa 647-labeled B220 (blue). Very few or CD8 cells contained green fluorescent particles (arrowheads). he asterisks highlight the magnified areas shown in Fig. 5. indicates marginal zone;, cell area. (Scale bars: 100 m.) 2 of 6

3 F4/80 SIGNR1 RP CD169 SIGNR1 RP Fig. S3. Accumulation of YG-PG in RP and macrophages. hirty minutes after inoculation of YG-PS intravenously, BALB/c spleen sections were stained with -F4/80 (Left)or -CD169 (Right), followed by Alexa 555 anti-rat IgG (red), and anti-signr1 Alexa 647 (blue). indicates cell area;, marginal zone; RP, red pulp. Asterisks highlight magnified regions shown in Fig. 5. (Scale bar: 100 m.) 3of6

4 A Control 12 h 48 h B CD40 Isotype PBS Poly IC 6 h Poly IC 12 h LPS 6 h LPS 12 h CD86 Fig. S4. LPS inoculation induces in situ disappearance of Langerin. (A) As in Fig. 5, mice were inoculated intraperitoneally with PBS (Left)or25 g of LPS (Center and Right) 30 min after injecting YG-PS intravenously. Spleen sections were analyzed 12 h (Left and Center)or48h(Right) after microbial agonist inoculation. In addition to the green label from fluorescent YG particles, primarily in the RP and, the sections were stained for Langerin (red) and B220 (blue). (Scale bar: 200 m.) (B) Splenic CD8 DCs up-regulate CD40 and CD86 after microbial agonist inoculation. Mice were inoculated with YG-PS 30 min before the inoculation of 50 g of poly(ic) or 25 g of LPS intraperitoneally. Animals were killed 6 or 12 h later and analyzed for cell surface expression of CD40 (Left) and CD86 (Right) on CD8 DCs. 4of6

5 able S1. Antibodies used for multicolor flow cytometry Antibody Fluorochrome Clone Specificity B220 FIC RA3 6B2 CD45R CD3 Pacific Blue; Alexa A2 cell lineage CD8 PerCP Cy CD8 CD11b PerCP Cy5.5 and APC-Alexa 750 M1/70 M 2 PE HL3 x 2 integrin APC-Alexa 750 N418 x 2 integrin CD115 PE AFS98 CSF-1R CD19 PE-Cy7 1D3 B cells CD49b FIC; PE-Cy7 DX5 CD49b/pan-NK cells F4/80 PerCP Cy5.5 BM8 F4/80/pan-macrophages marker Ly6G FIC 1A8 Ly6G / granulocytes PDCA-1 PE JF05 1C2.4.1 PDCA-1 / Plamacytoid DCs Streptavidin PE; APC Biotin er119 PE-Cy7 ER-119 Erythrocytes All antibodies are from BD Biosciences or ebiosciences, except for PDCA-1 (Miltenyi Biotec). PE, phycoerythrin. 5of6

6 able S2. Antibodies used for Immunofluorescence Antibody Fluorochrome Specificity Clone Isotype Final concentration Source Secondary antibody hird antibody B220 Alexa 647 CD45R RA3 6B2 Rat IgG2a 4 g/ml BD - - CD8 Biotin CD Rat IgG2b 3 g/ml Our Lab Sva Alexa CD8 Purified CD Rat IgG2b 3 g/ml Our Lab -Rat Alexa Purified x 2 integrin HL3 Ar Hamster 3 g/ml BD - Hamster FIC - FIC Alexa 488 Purified x 2 integrin HL3 Ar Hamster 3 g/ml BD - Hamster Sva Alexa 555 Biotin CD169 FIC Sialoadhesin MOMA-1 Rat IgG2a 1 g/ml AbD Serotec - FIC Alexa CD169 Purified Sialoadhesin 3D6.112 Rat IgG2a 1.5 g/ml AbD Serotec - Rat Alexa 555 CD207 Purified Langerin L31 Rat IgG2a 3 g/ml Our Lab - Rat Alexa or Alexa 555 CD209b Alexa 647 SIGNR1 22D1 Ar Hamster 3 g/ml Our Lab - - F4/80 FIC Macrophages CI:A3 1 Rat IgG2b 0.3 g/ml AbD Serotec - FIC Alexa F4/80 Purified Macrophages CI:A3 1 Rat IgG2b 1.5 g/ml AbD Serotec - Rat Alexa DEC205-OLLAS Purified DEC205 Mouse IgG1 6 g/ml Our Lab - OLLAS Biotine Sva Alexa 555 Molecular Probes, Invitrogen. Jackson Immunoresearch. Our lab. 6of6

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