mmcp6  (R&D Systems)

Journal: International Journal of Molecular Sciences

Article Title: Stress Affects Mast Cell Proteases in Murine Skin in a Model of Atopic Dermatitis-like Allergic Inflammation

doi: 10.3390/ijms25115738

Figure Lengend Snippet: Stress and stress mediator effects on mMCP protein and mRNA expression in MCs. Data represent the mean values of the results from 5 animals ( n = 5). ** = p ≤ 0.01; * = p ≤ 0.05. Immunohistomorphometric analysis was performed in 10 microscopic fields per animal on 14 µm thick skin cryosections for ( a , d ) mMCP4+ and mMCP6+ MCs (expressed in % of total number of FITC-Avidin+ MCs). FITC-Avidin is used as a specific MC marker in skin. ( c , f ) High- (black bar) and low-intensity (gray bar) staining of mMCP4+ and mMCP6+ MCs (sums up to % of mMCP+ MCs of the respective group given in ( a , d )). ( b , e ) Representative photomicrographs of an immunohistochemical triple staining for mMCP4 and mMCP6 (red), MCs (FITC-labeled avidin, green), and cell nuclei (DAPI, blue), scale bar = 10 µm. Abbr.: AlD = atopic dermatitis-like allergic inflammation; anti-BDNF = antibody against BDNF; anti-NGF = antibody against NGF; BDNF = brain-derived neurotrophic factor; MC = mast cell; mMCP = murine mast cell protease; NGF = nerve growth factor; NiS = noise induced stress.

Article Snippet: The primary antibodies used in this study are c-Kit (1:100, Acris Antibodies, Herford, Germany), mMCP4 (1:400, Biozol, Eching, Germany), and mMCP6 (1:800, AF3736, R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Avidin-Biotin Assay, Marker, Staining, Immunohistochemical staining, Labeling, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Stress Affects Mast Cell Proteases in Murine Skin in a Model of Atopic Dermatitis-like Allergic Inflammation

doi: 10.3390/ijms25115738

Figure Lengend Snippet: SP induces mMCP4 mRNA in the presence of Chrna7 in cultured MCs. ( a , b ) qRTPCR analysis of mMCP4 and mMCP6 mRNA expression in primary cultures of peritoneal MCs from Wt and α7-KO mice. Individual treatments are mentioned at the X axis of the graphs. Gene expressions were normalized to the housekeeping gene TBP. Data represent the averages of at least 3 different experiments. * = p ≤ 0.05. Abbr.: α7-KO = Chrna7 knockout; mMCP = murine mast cell protease; SLURP1 = Ly-6/uPAR-related protein 1; SP = substance P; Wt = wild type.

Article Snippet: The primary antibodies used in this study are c-Kit (1:100, Acris Antibodies, Herford, Germany), mMCP4 (1:400, Biozol, Eching, Germany), and mMCP6 (1:800, AF3736, R&D Systems, Minneapolis, MN, USA).

Techniques: Cell Culture, Expressing, Knock-Out

Journal: International Journal of Molecular Sciences

Article Title: Stress Affects Mast Cell Proteases in Murine Skin in a Model of Atopic Dermatitis-like Allergic Inflammation

doi: 10.3390/ijms25115738

Figure Lengend Snippet: Primers and probes used for qRTPCR.

Article Snippet: The primary antibodies used in this study are c-Kit (1:100, Acris Antibodies, Herford, Germany), mMCP4 (1:400, Biozol, Eching, Germany), and mMCP6 (1:800, AF3736, R&D Systems, Minneapolis, MN, USA).

Techniques: Sequencing

Journal: Cells

Article Title: The Role of Galectin-9 as Mediator of Atopic Dermatitis: Effect on Keratinocytes

doi: 10.3390/cells10040947

Figure Lengend Snippet: Inflammatory response in the skin. Intense influx of eosinophils ( b , d ; black arrows) and activated mast cells ( d ; white arrows) were observed in the ( b ) dermis of AD group in comparison to the ( a ) SHAM. Mast cells in ( d ) showed a weaker staining compared to the cell in ( c ) related to their greater activation with the release of cytoplasmic granules. Stain: Diff-Quick. ( e , f ) Quantification of intact (IMCs) and degranulated mast cells (DMCs) in the dermis. IMCs were characterized by metachromatic cytoplasmic granules, while DMCs by the exocytosis of granules in the dermis. Data represent mean ± SEM of the number of cells per mm 2 ( n = 5 animals/group). *** p < 0.001 vs. Naïve/Degranulated MCs; ### p < 0.001 vs. Sham/Degranulated MCs (ANOVA, Bonferroni post-test). ( g , h ) Immunofluorescence double staining for mouse mast cell protease 6 (mMCP6) or eosinophil peroxidase (EPX) and Gal-9 in AD skin. mMCP6 and EPX are colocalized with Gal-9 in the cytosol of mast cells ( g ) and eosinophils ( h ). DAPI was used as a nuclear counterstain. Scale bars: 25 µm ( a,b ); 10 µm ( c,d,g,h ); 5 µm ( e ).

Article Snippet: Colocalization of Gal-9 and specific markers for mast cells and eosinophils in mouse skin was performed through incubation of sections with polyclonal antibody goat anti-mMCP6 (mouse mast cell protease 6; R&D Systems, Minneapolis, MN, EUA) or anti-EPX (eosinophil peroxidase; Santa Cruz Biotechnology, Dallas, TX, USA), diluted 1:300 in PBS 1% BSA for 16–18 h at 4 °C.

Techniques: Comparison, Staining, Activation Assay, Diff-Quik, Immunofluorescence, Double Staining

Journal: The Journal of Clinical Investigation

Article Title: Mast cell hyperactivity underpins the development of oxygen-induced retinopathy

doi: 10.1172/JCI89893

Figure Lengend Snippet: ( A ) mMCP6 ELISA was performed using serum from the OIR model and age-matched naive WT pups on P17. n = 8 in each group. * P < 0.05; ** P < 0.01 versus saline-injected mice, Dunnett’s test. ( B ) mMCP6 immunohistochemical analysis was performed on skin sections of WT mice on P7 and P17 and on BMCMCs cultured for 5 weeks. Results are representative of 3 independent experiments. Arrowheads indicate mast cells in the skin. On P17, mast cells were degranulated and mMCP6 positivity was not identified. ( C ) mMCP6 ELISA was performed using mouse serum obtained at 6 hours after treatment with cromolyn on P12. n = 8 in each group. * P < 0.05 versus control mice, Mann-Whitney U test. ( D ) Schematic of the study design. Pups were i.p. injected with 20 μl NM or anti-mMCP6 mAbs every day from P12 to P17. Animals were sacrificed, and eyes were enucleated on P17. ( E ) Quantification of neovascular nuclei in mice treated with NM and anti-mMCP6 mAbs. n = 9 in each group. ** P < 0.01 versus saline-injected mice, Dunnett’s test. ( F ) Quantification of neovascular nuclei in mice injected with recombinant mMCP6. n = 9 in each group. * P < 0.05; ** P < 0.01 versus Kit Wsh/Wsh mice treated with vehicle alone, Dunnett’s test. ( G ) OIR was not induced by reconstitution of Cpa3 Cre/+ mice with mMCP6-deficient BMCMCs. n = 8 in each group. ** P < 0.01 versus saline treatment, Dunnett’s test. ( H ) Retinal neovascularization in relative hypoxic condition in PAR2-deficient mice. n = 8 in each group. Scale bars: 50 μm ( B , skins); 20 μm ( B , BMCMC). Results are shown as mean ± SEM of values determined from 3 to 4 independent experiments ( A , C , E – H ).

Article Snippet: We injected 20 μl of a mast cell stabilizer, cromolyn ( ) (50 mg/kg, Sigma-Aldrich), a specific inhibitor of tryptase NM ( ) (1 mg/kg, Torii Pharmaceutical Co.), or anti-mMCP6 mAbs (15 μg/kg, R&D Systems, catalog MAB4288, clone 286828) i.p. into pups once a day.

Techniques: Enzyme-linked Immunosorbent Assay, Saline, Injection, Immunohistochemical staining, Cell Culture, Control, MANN-WHITNEY, Recombinant

Journal: The Journal of Clinical Investigation

Article Title: Mast cell hyperactivity underpins the development of oxygen-induced retinopathy

doi: 10.1172/JCI89893

Figure Lengend Snippet: ( A ) Mcp1 mRNA expression was significantly increased in OIR mice. This was suppressed by administration of cromolyn, but administration of PBS alone did not have any effect. Other angiogenic factors, Vegf and Fgf , were upregulated in OIR mice and decreased by cromolyn treatment. On P11 and P12, WT mice were injected with PBS or cromolyn, and eyes were collected at 6 hours after the second administration on P12. n = 8 in each group. ** P < 0.01 versus PBS-injected mice, Dunnett’s test. ( B ) Addition of recombinant mMCP6 into the culture of primary retinal endothelial cells induced Mcp1 , Vegf , Fgf , and Hgf gene expression. n = 8 in each group. * P < 0.05; ** P < 0.01 versus vehicle treatment, Mann-Whitney U test. ( C ) Schematic of Mcp1 gene–silencing experiments. Intravitreal injection of MCP1 siRNA effectively suppressed retinal MCP1 expression. n = 8 in each group. ** P < 0.01 versus control RNA-injected mice, Mann-Whitney U test. ( D ) Abnormal angiogenesis, following relative hypoxia, was suppressed by the specific inhibition of MCP1 in the retina. Neovascularization area (%) was quantified in whole-mount specimens. n = 8 in each group. ** P < 0.01 versus PBS-injected mice, Mann-Whitney U test. ( E ) Relative hypoxia induced the formation of new abnormal blood vessels (white areas) in WT mice ( n = 14), but not in CCR2-deficent mice ( n = 8). ** P < 0.01 versus WT mice, Mann-Whitney U test. ( F ) Addition of recombinant mMCP6 into the culture of primary retinal endothelial cells induced typical tube formation. The effect of mMCP6 was suppressed by addition of anti-MCP1 mAbs. n = 4 in each group. * P < 0.05 versus rat isotype mAb, Dunnett’s test. ( G ) Addition of recombinant mouse MCP1 (10 ng/ml) into the culture of primary retinal endothelial cells induced typical vascular tube formation. n = 4 in each group. ** P < 0.01 versus vehicle control, Mann-Whitney U test. Scale bars: 500 μm ( D , E ); 100 μm ( F , G ). All results are shown as mean ± SEM of values determined from 3 to 4 independent experiments.

Article Snippet: We injected 20 μl of a mast cell stabilizer, cromolyn ( ) (50 mg/kg, Sigma-Aldrich), a specific inhibitor of tryptase NM ( ) (1 mg/kg, Torii Pharmaceutical Co.), or anti-mMCP6 mAbs (15 μg/kg, R&D Systems, catalog MAB4288, clone 286828) i.p. into pups once a day.

Techniques: Expressing, Injection, Recombinant, Gene Expression, MANN-WHITNEY, Control, Inhibition