Human DC-SIGN/CD209 Antibody

Detection of DC-SIGN in Human DC-SIGN Transfected 3T3 Mouse Cell Line by Flow Cytometry.

Human DC-SIGN and DC-SIGN2 transfected 3T3 mouse embryonic fibroblast cell line were stained with Mouse Anti-Human DC-SIGN Monoclonal Antibody (Catalog # MAB161, filled histograms) or isotype control antibody (Catalog # MAB0041, open histogram), followed by Phycoerythrin-conjugated Anti-Mouse IgG F(ab')₂Secondary Antibody (Catalog # F0102B).

Detection of DC-SIGN in Human Monocyte Derived Dendritic Cells by Flow Cytometry.

Human monocyte derived dendritic cells were stained with Mouse Anti-Human DC-SIGN Monoclonal Antibody (Catalog # MAB161) followed by PE-conjugated anti-mouse IgG (Catalog # F0102B) and Anti-Human B7-2/CD86 Fluorescein-conjugated Monoclonal Antibody (Catalog # FAB141F). Quadrant markers were set based on control antibody staining (Catalog # MAB0041).

Detection of Human DC-SIGN/CD209 by Flow Cytometry

Infectivity of KSHV is enhanced in the presence of DC-SIGN and DC-SIGNR.A) 293T cells were transfected with empty pcDNA3 vector or expression constructs for DC-SIGN or DC-SIGNR. After 24 hours, cells were infected with 20 µl Bac16 deltaK3 deltaK5 or left uninfected as controls. Cells were harvested after additional 24 hours and surface stained with a DC-SIGN/R antibody (H-200) and analyzed by flow cytometry. Top three panels show transfected cells stained for DC-SIGN/R followed by PE- (DC-SIGN), FITC- (DC-SIGNR) or both (vector) conjugated secondary antibodies. Bottom panels shows KSHV infection of 293T cells transiently expressing DC-SIGN or DC-SIGNR. B, left panels) 293 cell lines stably expressing a vector construct, DC-SIGN or DC-SIGNR were fluorescently stained for surface expression of DC-SIGN or DC-SIGNR. The mean channel fluorescence is indicated in the upper right hand corner. Open histograms – secondary antibody alone; shaded histograms – DC-SIGN or DC-SIGNR staining. B, right panel) 293 pcDNA3, DC-SIGN or DC-SIGNR stable cell lines were pre-incubated with a control antibody (anti-ICAM1, 7 µg/ml), with mannan (100 µg/ml) or a monoclonal antibody specific for DC-SIGN (MAB161; 7 µg/ml) for 30 minutes on ice. These cells were then infected with wild type KSHV (Bac16 or rKSHV.219) at an MOI of 0.01. After 72 hours cells were harvested and evaluated for infection by flow cytometry measuring GFP expression. Infection rates were normalized to 293 pcDNA3 cells treated with the control antibody. The fold increase in relative infectivity is indicated. Data are representative of four independent experiments with two performed in triplicate. C) 293 pcDNA3, DC-SIGN or DC-SIGNR stable lines were infected with 50 µl of concentrated Bac16 wildtype (wt), or mutants with deletion of K3 only ( deltaK3), K5 only ( deltaK5), or deletion of both K3 and K5 ( deltaK3 deltaK5) as indicated. At 72 hours post-infection, the cells were stained for surface expression of DC-SIGN, DC-SIGNR or MHC class I. GFP fluorescence was used as a marker for infection. MHC I staining is shown for infected 293 DC-SIGNR cells. Inset numbers indicate percentage of cells in each quadrant. Image collected and cropped by CiteAb from the following publication (https://dx.plos.org/10.1371/journal.pone.0058056), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Cynomolgus Monkey DC-SIGN/CD209 by Immunohistochemistry

Rapid induction and persistence of DC-SIGN, S100 and CD68 post-vaccination in lymphoid tissue.Galleries of representative fields of A DC-SIGN and B CD68 staining in MLN at 3, 7, 10, 21, 125 d.p.i. C Representative fields of DC-SIGN signal tracking through the spleen 3–125 d.p.i. maximal from d3 in red pulp (rp) and marginal zones (mg) D Representative fields of splenic S100 levels illustrate similar staining intensity 3–125 d.p.i. predominantly in the germinal centres and red pulp. Staining intensities scoring were; pale yellow (no staining, −); yellow (very low, +); dark yellow (low, ++), red (medium, +++) and magenta (high, ++++). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25162725), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of Cynomolgus Monkey DC-SIGN/CD209 by Immunohistochemistry

Rapid induction and persistence of DC-SIGN, S100 and CD68 post-vaccination in lymphoid tissue.Galleries of representative fields of A DC-SIGN and B CD68 staining in MLN at 3, 7, 10, 21, 125 d.p.i. C Representative fields of DC-SIGN signal tracking through the spleen 3–125 d.p.i. maximal from d3 in red pulp (rp) and marginal zones (mg) D Representative fields of splenic S100 levels illustrate similar staining intensity 3–125 d.p.i. predominantly in the germinal centres and red pulp. Staining intensities scoring were; pale yellow (no staining, −); yellow (very low, +); dark yellow (low, ++), red (medium, +++) and magenta (high, ++++). Image collected and cropped by CiteAb from the following publication (https://pubmed.ncbi.nlm.nih.gov/25162725), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of DC-SIGN/CD209 by Immunohistochemistry

Infection of DC-SIGN+ cells in the lung.(A) Infection of the DC-SIGNhi, DC-SIGNlo and DC-SIGN- cells in BAL from MV-infected macaques, determined by detection of EGFP in flow cytometry at day 2–5 d.p.i. Each dot represents an individual animal. Lines indicate geometric means. (B) Macroscopic images from EGFP+ lung slices collected 3 d.p.i., cultured for additional 3,5,7 or 10 days. (C) Phenotype of cells migrating from the ex vivo cultured lung slice, collected from supernatant after 5 days of culturing (D) Phenotype of EGFP+ cells collected from lung slice medium. (E-F) DC-SIGN expression on lung sections from uninfected macaques (E) or 2.d.p.i. (F) Asterisks indicate DC-SIGN reactivity. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/23227146), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of DC-SIGN/CD209 by Western Blot

K3 and K5 affect the stability of DC-SIGN and DC-SIGNR in a RING-CH domain-dependent mechanism.A) 293 cells stably expressing wild-type K3 or K5, or the RING-CH mutant of either viral protein were transiently transfected with 2 µg DC-SIGN or DC-SIGNR constructs. At ∼48 hpt,cells were lysed in RIPA buffer and 30 µg of normalized lysate were loaded per sample. Protein levels of DC-SIGN or DC-SIGNR were determined by WB, and then blots were reprobed for lamin B as a loading control. Data is representative of at least three independent experiments. B) THP-1 cell stably expressing the indicated K5 constructs or empty vector were stained for cell surface levels of DC-SIGN. Solid histogram, empty vector; grey histogram, K5 construct; dotted histogram, isotype control. C) The same THP-1 cell lines used in Panel B, were lysed and subjected to western blotting with either a DC-SIGN (H-200) or GAPDH (O411) antibody, as loading control. Data is representative of at least three independent experiments. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/23460925), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of DC-SIGN/CD209 by Immunocytochemistry/ Immunofluorescence

DC-SIGN is expressed by DCs and a subset of macrophages in lymph nodes.(A) TBLN cells were stained for DC-SIGN in combination with DC and macrophage markers and analyzed by flow cytometry. Gray areas show negative controls (DC-SIGN single staining). Percentages of positive cells expressing the markers are annotated in the upper right corner. (B) DC-SIGN expression of CD11c+ and CD83+ cells in TBLNs. (C-D) Dual immunofluorescence staining of DC-SIGN (green) and MAC387 (red) in lung sections 2 or 3 d.p.i. (C i-iv) and axillary lymphoid tissue 4 d.p.i. (D). Nuclei are stained blue with Hoechst. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/23227146), licensed under a CC-BY license. Not internally tested by R&D Systems.