cd11c + magnetic bead sorting  (STEMCELL Technologies Inc)

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Lkb1 deletion in CD11c + macrophages drives fibrosis and inflammation in CP. (A) Representative images of pancreatic H&E staining. Scale bar, 100 µm. n = 8. (B) Representative images of pancreatic Masson's staining. Scale bar, 100 µm. n = 8. (C) Representative immunohistochemistry (IHC) staining images of CD11c in pancreatic tissue. Scale bar, 50 µm. n = 8. (D,E) Representative immunofluorescence images of CD11c, Lkb1, and DAPI co‐staining in CP and normal pancreatic tissues. Scale bar, 50 µm. n = 8. (F) Western blot analysis of Lkb1 expression in pancreatic CD11c + cells at multiple time points after disease induction. n = 3. (G,H) Representative immunofluorescence images of CD11c, Lkb1, and DAPI co‐staining in pancreatic tissues from control and CP model WT mice, as well as in splenic tissues from control WT mice. Scale bar, 50 µm. n = 5. (I–L) Representative images of pancreatic H&E staining and Masson's staining. Scale bar, 100 µm. From left to right: n = 8; n = 8; n = 12; n = 10. (M) Representative immunofluorescence images of co‐staining for CD45, CK19, α‐Amylase, α‐SMA, and DAPI in the pancreas with (N,O) statistical analysis. Scale bar, 50 µm. n = 5. (P) Relative pancreatic weight (pancreas/body weight). From left to right: n = 8; n = 8; n = 12; n = 10. Data are means ± SEM. Unpaired Student's t ‐tests (A, B, C, E, H, N, O) were used to evaluate statistical significance. Data were analyzed using one‐way ANOVA (J, L, P) with the Tukey test.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: Staining, Immunohistochemistry, Immunofluorescence, Western Blot, Expressing, Control

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Lkb1 deletion in CD11c + cells modulates the inflammatory microenvironment of CP. (A) Overview of the CP mouse model and scRNA‐seq workflow. (B) UMAP plots illustrating distinct cell populations in the pancreas of CP mice. (C) UMAP plot displaying distinct cell populations in CP pancreatic tissues of Lkb1 f/f and CD11c Cre Lkb1 f/f mice. (D) Percentage of cells of different cell types in CP pancreatic tissues of Lkb1 f/f and CD11c Cre Lkb1 f/f mice. (E–G) Differences in the frequency of PSCs、T cells or macrophages as a percentage of total cells. (H) KEGG enrichment analysis of upregulated genes in PSCs. (I) UMAP plot of macrophage subclusters in CP pancreatic tissue. (J) Percentage of cells of different macrophage subclusters in macrophages of Lkb1 f/f and CD11c Cre Lkb1 f/f mice. (K) Trajectory plots display pseudo‐time trajectory analysis of various macrophage subpopulations derived from monocytes. (L) Violin plot showing the progression of cells from Lkb1 f/f and CD11c Cre Lkb1 f/f mice along the determined trajectory in pseudotime. (A–L) Lkb1 f/f ( n = 4) and CD11c Cre Lkb1 f/f ( n = 3) mice (each dot represents an animal). Data are means ± SEM. Unpaired Student's t ‐tests (E–G) were used to evaluate statistical significance.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: Derivative Assay

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Lkb1 deletion in CD11c + cells promotes an increase in CD11c + CD206 + macrophages in CP. (A,B) Representative flow cytometry plots and the absolute number of macrophages in the pancreas. From left to right: n = 5; n = 4. (C,D) Representative flow cytometry plots and the absolute number of CD11c + CD206 + macrophages in the pancreas. From left to right: n = 5; n = 4. (E–G) Representative IHC staining images and statistical analysis of CD11c ( n = 10; n = 8), F4/80 ( n = 12; n = 9), and CD206 ( n = 12; n = 8). Scale bar, 50 µm. (H,I) Representative immunofluorescence images of co‐staining for F4/80, CD206, CD11c, α‐SMA, and DAPI in the pancreas. Scale bar, 50 µm. n = 8. (J,K) Representative immunofluorescence images of CD68, CD206, CD11c, and DAPI co‐staining in pancreatic tissues of patients with clinical CP. Scale bar, 50 µm. n = 8. Data are means ± SEM. Unpaired Student's t ‐tests were used to evaluate statistical significance.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: Flow Cytometry, Immunohistochemistry, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Lkb1 deletion in CD11c + cells promotes CCL2 secretion and recruitment of macrophages. (A) UMAP plots showing the gene expression and distribution of CCL2 in the subpopulation. Lkb1 f/f ( n = 4) and CD11c Cre Lkb1 f/f ( n = 3) mice. (B,C) Violin plots showing the expression levels of CCL2 in CD11c + CD206 + macrophages and PSCs. Lkb1 f/f ( n = 4) and CD11c Cre Lkb1 f/f ( n = 3) mice. (D,E) Immunofluorescence images showing co‐staining of CD11c, CD206, CCL2, α‐SMA, and DAPI in mouse pancreatic tissue. Scale bar, 50 µm. From left to right: n = 6; n = 5. (F,G) Immunofluorescence images showing co‐staining of α‐SMA, CCL2, and DAPI in mouse pancreatic tissue. Scale bar, 50 µm. From left to right: n = 6; n = 5. (H,I) Immunofluorescence images showing co‐staining of CD11c, CD206, CCL2, α‐SMA, and DAPI in human pancreatic tissue. Scale bar, 50 µm. n = 8. (J) Statistical chart of correlation between CCL2 + cells and α‐SMA + cells. (K) Schematic representation of the experimental model using CCL2 inhibitors. (L) Representative images of pancreatic tissue stained with H&E staining and Masson's staining, and (M) pathological scores indicating statistical analysis. Scale bar, 100 µm. From left to right: n = 6; n = 7; n = 6; n = 6. (N) Representative flow cytometry plots and (O) absolute numbers of total macrophages. From left to right: n = 6; n = 6; n = 5; n = 6. (P) Representative flow cytometry plots and (Q) absolute numbers of CD11c + CD206 + macrophages. From left to right: n = 6; n = 6; n = 5; n = 6. Data are means ± SEM. Unpaired Student's t ‐tests (E,F,I) were used to evaluate statistical significance. Data were analyzed using one‐way ANOVA (M,O,Q) with the Tukey test.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: Gene Expression, Expressing, Immunofluorescence, Staining, Flow Cytometry

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Lkb1 regulates the expression of its target gene CCL2 in CD11c + CD206 + macrophages by inhibiting STAT3 phosphorylation. (A) Single‐cell regulatory network inference and clustering analysis showing the number of regulons, the number of TFs, and the number of target genes obtained by prediction. (B) Regulon activity values of subpopulations using a heatmap to demonstrate the heterogeneity and distribution characteristics of regulons in subpopulations. (C) WGCNA‐based gene co‐expression network analysis. (D) Volcano plot showing differentially expressed genes in CD11c + CD206 + macrophages. (E) KEGG enrichment analysis of upregulated differential genes in CD11c + CD206 + macrophages. (F) Schematic diagram of cell culture. (G) Western blot analysis of p‐STAT3 and t‐STAT3 in CD11c + CD206 + cells. (H) CCL2 secretion level in cell culture supernatants. (I) mRNA expression level of CCL2 . (A–E) Lkb1 f/f ( n = 4) and CD11c Cre Lkb1 f/f ( n = 3) mice, (F–I) n = 3. Data are means ± SEM. Data were analyzed using one‐way ANOVA (H,I) with the Tukey test.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: Expressing, Phospho-proteomics, Single Cell, Activity Assay, Cell Culture, Western Blot

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Lkb1 in CD11c + cells mediates the interaction between CD11c + CD206 + macrophages and PSCs through the OSM signaling pathway. (A,B) Multi‐subpopulation network diagrams showing the number of ligand‐receptor pairs and expression abundance or probability information in cellular subpopulation pairs. (C) Bar graphs showing the proportion of CD11c + CD206 + macrophage‐PSCs interacting with signaling pathways in the two groups. (D,E) Dot plots of the cell‐cell communication between PSCs and CD11c + CD206 + macrophages. (F) Network map of cellular communication at the level of the OSM signaling pathway in the CD11c Cre Lkb1 f/f group. (G) Heatmap showing the relative importance of each cell group calculated based on four network‐centered metrics of OSM signaling in CP mice of the CD11c Cre Lkb1 f/f group. (H) Bar plots showing the contribution of each ligand‐receptor pair to the OSM signaling pathway. Characterization maps of (I) OSM and (J) OSMR gene expression. (K) Representative immunofluorescence images of co‐staining for CD11c, F4/80, OSM, and DAPI in the pancreas with (L) statistical analysis. Scale bar, 50 µm. (M) Representative immunofluorescence images of co‐staining for OSMR, α‐SMA, and DAPI in the pancreas with (N) statistical analysis. Scale bar, 50 µm. (O) Representative immunofluorescence images of co‐staining for CD11c, CD68, OSM, OSMR, α‐SMA, and DAPI in human CP and normal pancreatic tissues with (P,Q) statistical analysis. Scale bar, 50 µm. Mountain range plots showing gene expression in PSCs associated with the (R) MAPK or (S) NF‐κB signaling pathways. (A–J,R,S) Lkb1 f/f ( n = 4) and CD11c Cre Lkb1 f/f ( n = 3) mice, K‐N) n = 5, (O–Q) n = 8. Data are means ± SEM. Unpaired Student's t ‐tests (L–Q) were used to evaluate statistical significance.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques: Expressing, Protein-Protein interactions, Gene Expression, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: Liver Kinase B1 in CD11c + Cells Inhibits Fibrosis in Chronic Pancreatitis via the Oncostatin M Signaling

doi: 10.1002/advs.75018

Figure Lengend Snippet: Mechanistic model of Lkb1 ’s role in CD11c + cells in chronic pancreatitis.

Article Snippet: Total RNA was extracted from the pancreatic tissue of mouse CP or CD11c magnetic bead‐sorted cells (Miltenyi Biotec, #130‐125‐835) using TRIzol reagent, following the manufacturer's guidelines.

Techniques:

Journal: The Journal of Infectious Diseases

Article Title: The Major Surface Glycoprotein of Pneumocystis murina Does Not Activate Dendritic Cells

doi: 10.1093/infdis/jiy342

Figure Lengend Snippet: Flow cytometry of dendritic cell activation by lipopolysaccharide (LPS) and major surface glycoprotein (Msg). Immature mouse dendritic cells were generated in vitro, using granulocyte-macrophage colony-stimulating factor, and were either unstimulated or stimulated with LPS (300 ng/mL), sodium dodecyl sulfate (SDS)–Msg (5 µg/mL), or lyticase-Msg (5 µg/mL), for 24 hours. Dendritic cells were identified on the basis of CD11c expression, and the surface expression of CD80, CD86, CD40, and major histocompatibility complex (MHC) class II were measured by flow cytometry. The expression of activation markers was analyzed by FlowJo and compared to expression for the untreated-dendritic cells. The light gray line represents untreated dendritic cells, and the black line represents the stimulated population. The x-axis is fluorescence intensity, and the y-axis is normalized to mode, using FlowJo software. LPS (top row) led to increased expression of activation markers, while neither Msg preparation led to increased expression of any activation marker. Results are for 1 representative experiment of 4 experiments, all with similar results.

Article Snippet: Cytokine Production Dendritic cells were positively selected by magnetic separation using CD11c magnetic-activated cell-sorting (MACS) beads (Milteny Biotec).

Techniques: Flow Cytometry, Activation Assay, Generated, In Vitro, Expressing, Fluorescence, Software, Marker

Journal: The Journal of Infectious Diseases

Article Title: The Major Surface Glycoprotein of Pneumocystis murina Does Not Activate Dendritic Cells

doi: 10.1093/infdis/jiy342

Figure Lengend Snippet: Cytokine production by dendritic cells following activation. Positively selected (CD11c+) immature dendritic cells (106 cells/well) were either unstimulated or stimulated with lipopolysaccharide (LPS; 300 ng/mL), crude Pneumocystis murina antigen (20 µg/mL), β-glucan (300 ng/mL), sodium dodecyl sulfate (SDS)–major surface glycoprotein (Msg; 5 µg/mL), or lyticase-Msg (5 µg/mL) for 48 hours. Supernatants were collected, and levels of 10 cytokines were analyzed as described in Methods. Significant increases were seen for multiple cytokines following stimulation with LPS and, to a lesser extent, with β-glucan but not with either of the Msg preparations. Graphed values are the average ± SD of 3 independent experiments. The ratio paired t test was used to compare each stimulated sample with the unstimulated control. *P < .05 and **P < .01. IL-1β, interleukin 1β; IL-2, interleukin 2; IL-4, interleukin 4; IL-5, interleukin 5; IL-6, interleukin 6; IL-10, interleukin 10; IL-12p70, interleukin 12p70; TNF-α, tumor necrosis factor α.

Article Snippet: Cytokine Production Dendritic cells were positively selected by magnetic separation using CD11c magnetic-activated cell-sorting (MACS) beads (Milteny Biotec).

Techniques: Activation Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin stabilization directs splenic DC progenitors towards CD8α+ DC development. (A) Intracellular β-catenin expression in naïve Ex3fl/fl and Ex3DC−/− splenic CD11c+ cells. (B) Intracellular β-catenin levels in splenic CD4+ T cells isolated from Ex3fl/fl and Ex3DC−/− mice. The data show results from an individual mouse that is representative of of at least 3 experiments with 3–5 mice per group. (C) Western blot analysis of β-catenin in bone marrow-derived DC from Ex3fl/fl and Ex3DC−/− mice following cytoplasmic (C) and nuclear (N) fractionation. Antibodies against PARP and Rab5 were used for nuclear and cytoplasmic loading controls, respectively. The data are from 1 independent trial. (D and E) Comparison pre-cDC populations from the (D) bone marrow and (E) spleen of Ex3fl/fl and Ex3DC−/− mice by flow cytometry. Numbers in representative plots represent percentages of relevant populations within the indicated gate. Bar graphs show mean percentages plus standard error (S.E.) of relevant populations. The data represent the combination of 2 independent experiments (n=10 mice per group). (F) Levels of splenic pre-CD8α+ DC, defined as CD11c+CD8α−B220−CD24+, in Ex3fl/fl and Ex3DC−/− mice by flow cytometry. The data are representative of 3 independent experiments, each involving 4–5 mice per group. **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Expressing, Isolation, Western Blot, Derivative Assay, Fractionation, Comparison, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin stabilization expands splenic CD8α+ and plasmacytoid DC populations. (A–G) Mature DC subset analysis of naïve Ex3fl/fl and Ex3DC−/− splenocytes by flow cytometry. (A and B) Percentage and total number of CD11c+ cells in Ex3fl/fl and Ex3DC−/− spleens. (C–E) Percentage and total number of (C and D) CD8α+ DC and (C and E) CD11b+ DC in naïve Ex3fl/fl and Ex3DC−/− spleens. The data are representative of at least 3 independent experiments (n=3–5 mice per group). (F and G) Percentage and total number of B220+PDCA-1+ plasmacytoid DC in naïve Ex3fl/fl and Ex3DC−/− spleens. (H and I) Mature DC subset analysis of naïve Ex3fl/fl and Ex3DC−/− lung tissue by flow cytometry. (H) Plots from representative mice and (I) percentage of CD103+CD11b− lung DC for multiple mice are shown. (J–L) Mature DC subset analysis of naïve Ex3fl/fl and Ex3DC−/− intestinal lamina propria by flow cytometry. (J) Plots from representative mice and percentages of (K) CD103+CD11b− and (L) CD103+CD11b+ intestinal DC for multiple mice are shown. Dots in relevant graphs represent results from individual mice. Bar graphs display means and standard errors of individual mice. The data are representative of at least 2 independent experiments (n=3–5 mice per group). *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin signaling controls Irf8 expression. (A) Semi-quantitative PCR analysis of Nfil3, Batf3, Id2, and Irf8 mRNA in CD11c+ splenocytes magnetically purified from naïve Ex3fl/fl and Ex3DC−/− mice. mRNA levels were normalized to GAPDH. The data are representative of 2 independent experiments (n=2–3 mice per group) (B) Representative flow cytometric plots of IRF8 expression by Ex3fl/fl and Ex3DC−/− CD8α− and CD8α+ splenic DC. (C) MFI of IRF8 within Ex3fl/fl and Ex3DC−/− CD8α− and CD8α+ DC and (D) the percent of CD8α+ DC expressing IRF8 are shown. Dots represent results from individual mice. The data are the combined results of 2 experiments, and the experiment was independently performed at least 3 times (n=4–5 mice per group). (E) Representative FACS plot of IRF4 expression and IRF4 MFI in Ex3fl/fl and Ex3DC−/− CD11c+ splenocytes. The data are representative of 3 independent experiments (n=4 mice per group). (F) Chromatin immunoprecipitation of naïve Ex3DC−/− Flt3L DC cultures with control IgG or β-catenin antibody followed by quantitative PCR to determine Irf8 promoter occupancy. DNA levels were normalized to 1% input chromatin. The data are representative of 2 independent experiments. (G) Quantitative PCR analysis of Axin2 and Irf8 gene expression in BMDC following 5 hr culture with DMSO or ICG-001. Fold change is relative to DMSO control. The data are from one independent trial. (H and I) Intracellular expression of IRF8 and β-catenin following ICG-001 treatment of BMDC (H) or MutuDC1940 cells (I). The data are representative of 2 (MutuDC1940 cells) and 4 (BMDC) independent experiments with 3 replicates per treatment per experiment. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Purification, Chromatin Immunoprecipitation, Control, Gene Expression

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: β-catenin stabilization enhances IL-12 production by CD8α+ DC. (A) IL-12p40 production by naïve Ex3fl/fl and Ex3DC−/− splenocytes stimulated in vitro with LPS, STAg, or media control measured by ELISA. (B) IL-12p40 production by splenic CD11c+ DC magnetically purified from Ex3fl/fl and Ex3DC−/− mice stimulated in vitro with LPS, STAg, or media control measured by ELISA. (C) IL-12p40 production by CD8α+ and CD8α− DC DC purified from naïve Ex3fl/fl and Ex3DC−/− splenocytes following in vitro stimulation with media or STAg for 48 hr measured by ELISA. (D) IL-12p40 secretion by Ex3fl/fl splenocytes pre-treated with ICG-001 for 5 hr and then stimulated overnight with LPS or STAg measured by ELISA. (E) IL-12p40 production by splenocytes (106) from Ex3DC−/− mice cultured for 5 hr with 5 μM ICG-001 or DMSO and then stimulated with media, LPS (100 ng/ml), or STAg (25 μg/ml) overnight. (F) IL-12p40 production by MutuDC1940 cells (105) pre-treated with 20 μM ICG-001 or DMSO for 2 hr and then stimulated with media or STAg (25 μg/ml) overnight. The data are representative of at least 3 (A, F) and 2 (B–E) independent experiments, each involving 3–5 mice per group, except (C), which used pooled samples from 3 mice per experiment. *, p<0.05; **, p<0.01; ***, p<0.001.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: In Vitro, Control, Enzyme-linked Immunosorbent Assay, Purification, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: β-Catenin Signaling Drives Differentiation and Proinflammatory Function of IRF8-Dependent Dendritic Cells

doi: 10.4049/jimmunol.1402453

Figure Lengend Snippet: Constitutive DC β-catenin signaling increases the proinflammatory cytokine response to Toxoplasma. (A) Survival of Ex3fl/fl and Ex3DC−/− mice following i.p. infection with Toxoplasma Type II strain ME49 (25 cysts) (n=4–6 mice per group). The data are representative of at least 3 experiments. (B) Quantitative PCR amplification of parasite (B1 gene) and host DNA (ASL gene) isolated from Ex3fl/fl and Ex3DC−/− spleens 9 days post-infection. Parasite load is displayed as a ratio of parasite genomes to host genomes (n=3–4 mice per group). (C) IL-12p40 production by CD11c+ DC magnetically separated from Day-6 post-infection Ex3fl/fl and Ex3DC−/− splenocytes and cultured overnight without additional stimulation (n=3 mice per group). (D) IL-12p70 production by bulk splenocytes from Day-6 post-infection Ex3fl/fl and Ex3DC−/− mice (n=3 mice per group). The data are representative of 2 independent experiments. (E) IL-12p40 and IFN-γ production by splenocytes from Day-10 post-infection Ex3fl/fl and Ex3DC−/− mice cultured for 72 hr without additional stimulation (n=3–5 mice per group). The data are representative of 3 independent experiments. (F) IL-12p40, IFN-γ, and TNF-α levels in serum collected from Day-9 post-infection Ex3fl/fl and Ex3DC−/− mice (n=3–5 mice per group). The data are representative of 2 independent experiments. The means and S.E. of individual mice are shown. *, p<0.05; **, p<0.01.

Article Snippet: A single round of positive selection using CD11c + magnetic bead sorting was performed for purification of total splenic DC from single cell suspensions (Stem Cell Technologies), while two-step magnetic bead sorting, with an initial negative selection to enrich for DC followed by CD8α + positive selection (Miltenyi Biotec), was performed to isolate CD8α + splenic DC.

Techniques: Infection, Real-time Polymerase Chain Reaction, Amplification, Isolation, Cell Culture

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Exosomes-like nanoparticles from intestinal mucosal cells carry prostaglandin E2 and suppress activation of liver natural killer T cells

doi: 10.4049/jimmunol.1203170

Figure Lengend Snippet: Ex vivo analysis of NKT cell responses to IDENs and α-GalCer. (A) Sorted NKT cells from mice treated with PBS or IDENs and CD11c+ DCs sorted from livers of naïve mice were co-cultured for 3 d in the presence of α-GalCer. Cell division was assessed by labeling the NKT cells with CFSE (5 μM). (B) Proliferation of NKT cells from naïve mice were cultured with liver DCs in the presence of IDENs or PBS for 3h, and then stimulated by α-GalCer. The proliferation of the NKT cells was determined after a 16-hour pulse with 3H-thymidine and the cultured cells subsequently harvested. (C) Cytokine production by liver NKT cells from naïve mice cultured with BMDCs in the presence of IDENs or PBS for 3h, and then stimulated by α-GalCer (100ng/mL) for 24h. (D) Cytokine production by NKT cells from naïve mice pretreated by IDENs or PBS for 3h, and then stimulated by α-GalCer-tetramer for 24h. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are representative of three experiments (A) or are the mean ± SEM of five experiments (B, C, D).

Article Snippet: NKT cells enriched by negative selection (4×10 5 ) were cultured with liver DCs (1×10 5 ) isolated by CD11c magnetic bead sorting (Miltenyi Biotec).

Techniques: Ex Vivo, Cell Culture, Labeling

Journal: Immunology

Article Title: Inverse relationship between dendritic cell CCR9 expression and maturation state

doi: 10.1111/j.1365-2567.2009.03043.x

Figure Lengend Snippet: CCR9 is moderately expressed on bone marrow (BM)-generated dendritic cells (DC). (a) BM cells (2 × 106) were cultured in 24-well plates for 1 day. Cells were removed from wells, labeled with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates, or with a control isotype (HIgG–PE and RIgG–FITC), and events were collected on a fluorescence-activated cell sorter (FACScan). (b) Day 5-harvested and purified BM-generated DC were subcultured at 1 × 106 cells/ml in the presence of control H2O (10 μl). DC were harvested the next day and labeled with monoclonal antibodies (mAbs), as shown. Dot-plots show basal levels of CCR9 expression on DC and are representative of more than six independent experiments. In experiments, the addition of control stimuli [H2O, MRS broth, Luria–Bertani broth or the cytosine–phosphate–guanosine (CpG) control] did not alter the expression of CCR9 on DC in comparison with CCR9 expressed on day 5-harvested DC.

Article Snippet: In most experiments, DC were further enriched using CD11c magnetic antibody cell sorting (MACS) beads and columns according to the miniMACS system procedure (Miltenyi Biotec, Auburn, CA).

Techniques: Generated, Cell Culture, Labeling, Fluorescence, Purification, Expressing, Comparison

Journal: Immunology

Article Title: Inverse relationship between dendritic cell CCR9 expression and maturation state

doi: 10.1111/j.1365-2567.2009.03043.x

Figure Lengend Snippet: Costimulatory molecule regulation upon exposure of dendritic cells (DC) to microbial stimuli. DC were purified and stimulated with cytosine–phosphate–guanosine (CpG) (3 μm), Lactobacillus plantarum [105 colony-forming units (CFU)/ml] or E. coli (105 CFU/ml) in 1 ml of RPMI-1640/10% fetal bovine serum (FBS) and supplements, overnight in a 37°, 5% CO2 humidified incubator. Treatments were added in 10-μl quantities. Cells were collected the next day and labeled with CD11c–phycoerythrin (PE) and with CD40– or CD86–fluorescein isothiocyanate (FITC) conjugates. Histogram overlays show an increase in costimulatory molecule expression above that of H2O stimulation. The results are representative of two to three independent experiments.

Article Snippet: In most experiments, DC were further enriched using CD11c magnetic antibody cell sorting (MACS) beads and columns according to the miniMACS system procedure (Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Labeling, Expressing

Journal: Immunology

Article Title: Inverse relationship between dendritic cell CCR9 expression and maturation state

doi: 10.1111/j.1365-2567.2009.03043.x

Figure Lengend Snippet: CCR9 on dendritic cells (DC) is up-regulated in response to maturation stimuli. Purified DC (1 × 106 cells/ml, 24-well plates) were stimulated with cytosine–phosphate–guanosine (CpG) (3 μm), Lactobacillus plantarum (105 CFU/ml), Escherichia coli (105 CFU/ml) or granulocyte–macrophage colony-stimulating factor (GM-CSF) (7 ng/ml) plus interleukin-4 (IL-4) (20 ng/ml), overnight in a 37°, 5% CO2 incubator. Cells were stained with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates or with relevant isotypes (HIgG–PE and RIgG–FITC). The isotype staining shown is representative of background fluorescence obtained with cells from each treatment. Other dot-plot panels show CCR9 expression when DC were treated with different stimuli or with the control (water). The results are representative of three to five independent experiments.

Article Snippet: In most experiments, DC were further enriched using CD11c magnetic antibody cell sorting (MACS) beads and columns according to the miniMACS system procedure (Miltenyi Biotec, Auburn, CA).

Techniques: Purification, Staining, Fluorescence, Expressing

Journal: Immunology

Article Title: Inverse relationship between dendritic cell CCR9 expression and maturation state

doi: 10.1111/j.1365-2567.2009.03043.x

Figure Lengend Snippet: CCR9 is moderately expressed on lamina propria dendritic cells (DC). Lamina propria mononuclear cells (LPMC) were recovered from colons of mice with colitis by collagenase digestion. Cells were purified using CD11c magnetic antibody cell sorting (MACS) beads, labeled with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates and further purified on a Becton Dickinson FACS Aria. CCR9+ (upper right) and CCR9− (upper left) DC are shown in the dot-plot quadrants.

Article Snippet: In most experiments, DC were further enriched using CD11c magnetic antibody cell sorting (MACS) beads and columns according to the miniMACS system procedure (Miltenyi Biotec, Auburn, CA).

Techniques: Purification, FACS, Labeling

Journal: Immunology

Article Title: Inverse relationship between dendritic cell CCR9 expression and maturation state

doi: 10.1111/j.1365-2567.2009.03043.x

Figure Lengend Snippet: CCR9 is moderately expressed on bone marrow (BM)-generated dendritic cells (DC). (a) BM cells (2 × 106) were cultured in 24-well plates for 1 day. Cells were removed from wells, labeled with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates, or with a control isotype (HIgG–PE and RIgG–FITC), and events were collected on a fluorescence-activated cell sorter (FACScan). (b) Day 5-harvested and purified BM-generated DC were subcultured at 1 × 106 cells/ml in the presence of control H2O (10 μl). DC were harvested the next day and labeled with monoclonal antibodies (mAbs), as shown. Dot-plots show basal levels of CCR9 expression on DC and are representative of more than six independent experiments. In experiments, the addition of control stimuli [H2O, MRS broth, Luria–Bertani broth or the cytosine–phosphate–guanosine (CpG) control] did not alter the expression of CCR9 on DC in comparison with CCR9 expressed on day 5-harvested DC.

Article Snippet: Cells were purified using CD11c magnetic antibody cell sorting (MACS) beads, labeled with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates and further purified on a Becton Dickinson FACS Aria.

Techniques: Generated, Cell Culture, Labeling, Control, Fluorescence, Purification, Bioprocessing, Expressing, Comparison

Journal: Immunology

Article Title: Inverse relationship between dendritic cell CCR9 expression and maturation state

doi: 10.1111/j.1365-2567.2009.03043.x

Figure Lengend Snippet: Costimulatory molecule regulation upon exposure of dendritic cells (DC) to microbial stimuli. DC were purified and stimulated with cytosine–phosphate–guanosine (CpG) (3 μm), Lactobacillus plantarum [105 colony-forming units (CFU)/ml] or E. coli (105 CFU/ml) in 1 ml of RPMI-1640/10% fetal bovine serum (FBS) and supplements, overnight in a 37°, 5% CO2 humidified incubator. Treatments were added in 10-μl quantities. Cells were collected the next day and labeled with CD11c–phycoerythrin (PE) and with CD40– or CD86–fluorescein isothiocyanate (FITC) conjugates. Histogram overlays show an increase in costimulatory molecule expression above that of H2O stimulation. The results are representative of two to three independent experiments.

Article Snippet: Cells were purified using CD11c magnetic antibody cell sorting (MACS) beads, labeled with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates and further purified on a Becton Dickinson FACS Aria.

Techniques: Purification, Labeling, Expressing

Journal: Immunology

Article Title: Inverse relationship between dendritic cell CCR9 expression and maturation state

doi: 10.1111/j.1365-2567.2009.03043.x

Figure Lengend Snippet: CCR9 on dendritic cells (DC) is up-regulated in response to maturation stimuli. Purified DC (1 × 106 cells/ml, 24-well plates) were stimulated with cytosine–phosphate–guanosine (CpG) (3 μm), Lactobacillus plantarum (105 CFU/ml), Escherichia coli (105 CFU/ml) or granulocyte–macrophage colony-stimulating factor (GM-CSF) (7 ng/ml) plus interleukin-4 (IL-4) (20 ng/ml), overnight in a 37°, 5% CO2 incubator. Cells were stained with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates or with relevant isotypes (HIgG–PE and RIgG–FITC). The isotype staining shown is representative of background fluorescence obtained with cells from each treatment. Other dot-plot panels show CCR9 expression when DC were treated with different stimuli or with the control (water). The results are representative of three to five independent experiments.

Article Snippet: Cells were purified using CD11c magnetic antibody cell sorting (MACS) beads, labeled with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates and further purified on a Becton Dickinson FACS Aria.

Techniques: Purification, Staining, Fluorescence, Expressing, Control

Journal: Immunology

Article Title: Inverse relationship between dendritic cell CCR9 expression and maturation state

doi: 10.1111/j.1365-2567.2009.03043.x

Figure Lengend Snippet: CCR9 is moderately expressed on lamina propria dendritic cells (DC). Lamina propria mononuclear cells (LPMC) were recovered from colons of mice with colitis by collagenase digestion. Cells were purified using CD11c magnetic antibody cell sorting (MACS) beads, labeled with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates and further purified on a Becton Dickinson FACS Aria. CCR9+ (upper right) and CCR9− (upper left) DC are shown in the dot-plot quadrants.

Article Snippet: Cells were purified using CD11c magnetic antibody cell sorting (MACS) beads, labeled with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates and further purified on a Becton Dickinson FACS Aria.

Techniques: Purification, FACS, Labeling