Journal: Scientific Reports

Article Title: Regulation of CCR7-dependent cell migration through CCR7 homodimer formation

doi: 10.1038/s41598-017-09113-4

Figure Lengend Snippet: CCR7 homodimers are dissociated by the CCR7 TM4-derived peptide. ( A ) The levels of bioluminescence signals are presented for HEK293T cells transfected with the indicated combinations of CCR7-CGLuc, CCR7-NGLuc, CGLuc and NGLuc plasmids (1.5 μg each). A representative experiment from at least three independent experiments is shown. Data represent mean ± SD of triplicate samples. *p < 0.05 by one-way ANOVA. ( B ) The levels of bioluminescence signals are shown for cells transfected with combinations of CCR7-CGLuc and CCR7-NGLuc in the presence of the CCR7 TM4 peptide or the shuffled peptide (15 μg/ml). Relative luminescence was obtained by normalizing the values against untransfected cells. A representative experiment from at least three independent experiments is shown. Data represent mean ± SD of triplicate samples. *p < 0.05 by Student’s t test. ( C ) The levels of CCR7 homodimer in parental H9 cells were measured by in situ PLA. CCR7-CCR1 heterodimer formation detected by anti-CCR7 mAb-PLA Plus and anti-CCR1 mAb -PLA Minus (left), and CCR7 homodimer formation by anti-CCR7 mAb-PLA Plus and -PLA Minus (right). A representative experiment from at least three independent experiments is shown with the mean number of PLA signals plotted on the vertical axis. *p < 0.05 by Mann-Whitney’s U test. ( D ) CCR7 (top) or CCR1 (bottom) homodimer formation after treatment with the CCR7 TM4 peptide (right panel) or the shuffled peptide (left panel). The number of PLA signals per cell was counted using the Duolink Image Tool software. A representative experiment from three independent experiments is shown with the mean number of PLA signals plotted on the vertical axis. *p < 0.05 by Mann-Whitney’s U test; NS, not significant. ( E ) CCR7 expression levels were evaluated in the presence (red open histogram) or absence (blue open histogram) of the CCR7 TM4 peptide (15 μg/ml) by flow cytometry using anti-human CCR7 antibody or isotype control antibody (gray-filled histogram). MFI is indicated on the histograms. A representative experiment from three independent experiments is shown.

Article Snippet: The cells were fixed with 4% PFA for 10 min, and stained with 7.5 μg/ml anti-human CCR7 mAb (MAB197; R&D Systems) or anti-human CCR1 mAb (clone 53504; R&D Systems) conjugated with either the PLA Minus or PLA Plus probes generated with Duolink II Probemaker (Sigma).

Techniques: Derivative Assay, Transfection, In Situ, Software, Expressing, Flow Cytometry, Control

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in normal, herpes simplex virus‐inoculated, but asymptomatic healthy (BDN) and Behçet's disease (BD) mice. Peripheral blood mononuclear cells (PBMC) and lymph node cells were isolated from normal, BDN and BD mice. Frequencies of surface CCR1 + cells (a) and cytoplasm CCR1 + cells (b) of whole leukocytes were analyzed by FACS. Representative flow cytometric histograms of surface CCR1 + PBMC and lymph node cells are shown (c). (d) Protein expression of CCR1 by Western blot, (e) Expression ratio of CCR1 to GAPDH.

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques: Virus, Isolation, Expressing, Western Blot

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in cytokine‐treated normal and Behçet's disease (BD) mice. Interleukin‐10 (IL‐10) (25, 50 and 100 pg) and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (50, 100 and 200 pg) were intraperitoneally injected into normal mice for five consecutive days and peripheral blood mononuclear cells (PBMC) were isolated. Frequencies of CCR1 + cells were analyzed by FACS for expression on the surface and in the cytoplasm of mice after treatment with IL‐10 (a) or GM‐CSF (b). IL‐10 (50 pg) and GM‐CSF (200 pg) were intraperitoneally injected into BD mice for five consecutive days. PBMC and lymph node cells were isolated. Frequencies of surface CCR1 + (c) and cytoplasm CCR1 + (d) PBMC and lymph node cells derived from IL‐10‐ and GM‐CSF‐treated BD mice were analyzed by FACS.

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques: Injection, Isolation, Expressing, Derivative Assay

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in CCL3‐treated normal mice. CCL3 (50 pg and 100 pg) was intraperitoneally injected into normal mice for five consecutive days. Peripheral blood mononuclear cells (PBMC) and lymph node cells were then isolated. Frequencies of CCR1 + cells were analyzed by FACS on the surface (a) and in the cytoplasm (b) of PBMC and lymph node cells. Representative flow cytometric histograms of CCR1 + cells on the surface (a) and in the cytoplasm (b) of PBMC are shown in (c).

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques: Injection, Isolation

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in CCL3‐ and BX471‐treated Behçet's disease (BD) mice. CCL3 and BX471 were used to treat BD mice and frequencies of CCR1 + cells were analyzed by FACS on the surface (a) and in the cytoplasm (b) of peripheral blood mononuclear cells (PBMC) and lymph node cells. Frequencies of CCR1 + cells in normal mice after treatment with BX471 are shown in (c) and (d).

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques:

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in colchicine (Col) and pentoxifylline (Pento) ‐treated normal and Behçet's disease (BD) mice. Colchicine (2 μg/mouse/day) and pentoxifylline (400 μg/mouse/day) were used to treat mice. Frequencies of surface and cytoplasm CCR1 + peripheral blood mononuclear cells (PBMC) and lymph node cells were analyzed in normal mice (a, b) and BD mice (c, d) by FACS. (e) Representative flow cytometric histogram of surface and cytoplasm CCR1 + PBMC following colchicine or pentoxifylline treatment of BD mice.

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques:

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in anti‐CCL3 antibody‐treated normal mice. Anti‐CCL3 antibody (1, 2, 20, 50 and 200 ng) was intraperitoneally injected into normal mice for five consecutive days. Peripheral blood mononuclear cells (PBMC) and lymph node cells were isolated. Frequencies of surface CCR1 + (a) and cytoplasm CCR1 + (b) PBMC and lymph node cells were analyzed by FACS.

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques: Injection, Isolation

Journal: Immunology

Article Title: The role of CCR1 and therapeutic effects of anti‐CCL3 antibody in herpes simplex virus‐induced Behçet's disease mouse model

doi: 10.1111/imm.13102

Figure Lengend Snippet: Frequencies of CCR1 + cells in anti‐CCL3 antibody‐treated Behçet's disease (BD) mice. Anti‐CCL3 antibody 20 ng was intraperitoneally injected into BD mice for five consecutive days. Peripheral blood mononuclear cells (PBMC) and lymph node cells were then isolated. Frequencies of surface CCR1 + (a) and cytoplasm CCR1 + (b) PBMC and lymph node cells were analyzed by FACS. (c) The changes in disease severity score after CCL3 or anti‐CCL3 antibody treatment of BD mice. (d) Symptoms of BD mice after CCL3 or anti‐CCL3 antibody treatment.

Article Snippet: Erythrocytes were eliminated with ammonium–chloride–potassium solution and washed with phosphate‐buffered saline (PBS) followed by surface staining of 1 × 10 6 cells with mouse CCR1 allophycocyanin‐conjugated antibody (cat# FAB5986A; R&D Systems, Minneapolis, MN) for 30 min at 4° in the dark.

Techniques: Injection, Isolation