Journal: JCI Insight

Article Title: Dendritic cell immunoreceptor drives atopic dermatitis by modulating oxidized CaMKII-involved mast cell activation

doi: 10.1172/jci.insight.152559

Figure Lengend Snippet: ( A and B ) Representative immunofluorescence images of dorsal skin sections and fluorescence analysis of DCIR staining in the skin tissues of patients with AD and controls ( n = 8). Scale bar: 100 μm. ( C ) Quantification analysis of DCIR + tryptase + cells in the lesion skin of patients with AD and controls. ( D ) Flow cytometry analysis of DCIR expression in human mast cell line cKit + FcεRI + LAD2 cells. ( E ) Scheme of experimental protocol for the direct bindings of human recombinant DCIR (hrDCIR) to BSA, CRE, and Man-BSA. ( F ) Direct binding of different doses of hrDCIR (0–5.0 μg/mL) to BSA, CRE, or Man-BSA ( n = 3). ( G ) Representative immunofluorescence images of FITC-CRE uptake by LAD2. Scale bar: 15 μm. ( H and I ) Flow cytometry analysis ( H ) and quantification ( I ) of FITC-CRE uptake at different doses (1–500 ng/mL) by LAD2 cells ( n = 3). ( J ) Inhibition of FITC-CRE uptake in LAD2 cells pretreated with DCIR neutralizing antibody (α-DCIR) or IgG isotype ( n = 4). Data represent mean ± SEM of 2 independent experiments. Data in B , C , and I were compared using a 2-tailed Student’s t test. Data in F and J were compared by 2-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The sections were then incubated with the primary antibodies against mouse Tryptase (AF1937, R&D system), DCIR (MAB2617, R&D), or mouse IgG1 overnight at 4°C.

Techniques: Immunofluorescence, Fluorescence, Staining, Flow Cytometry, Expressing, Recombinant, Binding Assay, Inhibition

Journal: JCI Insight

Article Title: Dendritic cell immunoreceptor drives atopic dermatitis by modulating oxidized CaMKII-involved mast cell activation

doi: 10.1172/jci.insight.152559

Figure Lengend Snippet: ( A ) Representative skin images and EASI scores of PBS- and CRE-treated WT and DCIR –/– mice. ( B ) Representative H&E staining and epidermal thickness (μm) of skin tissues of PBS- and CRE-treated WT and DCIR –/– mice. ( C ) Representative Toluidine blue staining and quantification of cells with positive staining for Toluidine blue of skin tissue sections of PBS- and CRE-treated WT and DCIR –/– mice. Scale bar: 100 μm. Arrows represent mast cells. ( D ) Serum levels of specific IgE and IgG1 to CRE. ( E ) Quantitative PCR analysis of IL-4, IL-13, IL-33, and TNF-α expression in the skin tissues of PBS- and CRE-treated WT and DCIR –/– mice. Each circle represents 1 mouse. n = 8. Data represent mean ± SEM of 2 independent experiments. Data were compared by 2-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The sections were then incubated with the primary antibodies against mouse Tryptase (AF1937, R&D system), DCIR (MAB2617, R&D), or mouse IgG1 overnight at 4°C.

Techniques: Staining, Real-time Polymerase Chain Reaction, Expressing

Journal: JCI Insight

Article Title: Dendritic cell immunoreceptor drives atopic dermatitis by modulating oxidized CaMKII-involved mast cell activation

doi: 10.1172/jci.insight.152559

Figure Lengend Snippet: ( A ) Scheme of experimental protocol of i.v. transfer of DCIR + versus DCIR – mast cells into Kit W-sh/W-sh mice for the generation of AD mouse model. ( B ) Representative Toluidine blue staining and quantification of cells with positive staining for Toluidine blue of skin tissue sections of Kit W-sh/W-sh mice with DCIR + or DCIR – mast cells ( n = 9). ( C ) Representative immunofluorescence images of mast cells with (yellow) or without (blue) DCIR expression. ( D ) Representative skin images and EASI scores of Kit W-sh/W-sh mice with DCIR + or DCIR – mast cells ( n = 8). ( E ) Representative H&E staining and epidermal thickness (μm) of skin tissues of Kit W-sh/W-sh mice with DCIR + or DCIR – mast cells ( n = 8). Scale bar: 100 μm. Arrows represent mast cells. ( F ) Serum levels of specific IgE and IgG1 to CRE ( n = 8). ( G ) Quantitative PCR analysis of IL-4, IL-13, IL-33, and TNF-α expression in the skin tissues of Kit W-sh/W-sh mice with DCIR + or DCIR – mast cells. Each circle represents 1 mouse ( n = 8). Data represent mean ± SEM of 2 independent experiments. Data were compared by 2-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: The sections were then incubated with the primary antibodies against mouse Tryptase (AF1937, R&D system), DCIR (MAB2617, R&D), or mouse IgG1 overnight at 4°C.

Techniques: Staining, Immunofluorescence, Expressing, Real-time Polymerase Chain Reaction

Journal: Journal of cell science

Article Title: Podosomes of dendritic cells facilitate antigen sampling

doi: 10.1242/jcs.141226

Figure Lengend Snippet: (A–B) Confocal images of dendritic cells cultured on glass (A) and on filters with 1 μm pore size (B). Actin was labeled with phalloidin-Alexa fluor 546 (Phal; magenta) and clathrin was visualized by immunostaining (AB; green). The filters were impregnated with Alexa fluor 633-labeled gelatin (Filter; grey). The yellow line indicates the position of the orthogonal view. Yellow arrow heads indicate randomly selected actin-rich cores. The red arrow head indicates the approximate filter surface. (C) Quantification of the fraction of clathrin positive actin cores from panels A–B. (D–F) Same as panels A–C, but now with immunostaining for DC-SIGN, DCIR, Dectin-1, CD206 and CD71. Error bars show the spread of data for multiple cells from at least two independent experiments. Scale bars, 2 μm.

Article Snippet: The following primary antibodies were used for the immunofluorescence: mouse anti-vinculin (V9131, Sigma) at 1:200 dilution (v/v), mouse anti-talin (T3287, Sigma) at 1:100 (v/v), mouse anti-paxillin (349 | MAB3060, Sigma) at 1:100 (v/v), mouse anti-CD11b (ITGAM or Bear-1) (IM2581, Coulter) at 1:200 (v/v), mouse anti-CD29 (or ITGB1) (clone ts2/16) at 1:200 (v/v), mouse anti-MMP-14 (MAB3328, Bio Connect / MilliPore) at 1:100 (v/v), rat anti-tubulin (ab6161, Abcam) at 1:500 (v/v), mouse anti-human CD209 (or DC-SIGN) (551186, BD Bioscience) at 1:200 (v/v), mouse anti-human DCIR (DDX0180, Dendritics) at 1:200 (v/v), mouse anti-human Dectin-1 (MAB1859, R&D Systems) at 1:100 (v/v), mouse anti-human CD206 (555953, BD Biosciences) at 1:250 (v/v), mouse anti-human CD71 (347510, SALK) at 1:50 (v/v), mouse anti-clathrin light chain (C1985, Sigma) at 1:100 (v/v) and mouse anti-human MHC class II (clone Q5/13 ( Quaranta et al., 1980 )) at 20 μg ml −1 .

Techniques: Cell Culture, Labeling, Immunostaining