Human ChemR23 Antibody

Detection of ChemR23/CMKLR1 by Immunocytochemistry/ Immunofluorescence

Non-genomic modulation of ChemR23 expression on neutrophils (A,B) ChemR23 expression on human leukocytes was determined by flow cytometry on vehicle and TNF alpha (10 ng/ml; 20 min)-stimulated cells. Representative histograms shown in A. (C) Neutrophils were treated with vehicle, pro-inflammatory (TNF alpha ; fMLF, 1 μM; IL-8, 100 ng/ml) or anti-inflammatory mediators (annexin A1, 10 nM; alpha MSH, 10 nM; C15, 10 pM) followed by staining for ChemR23. ChemR23 expression was also assessed on isolated human neutrophils before and after flow over activated endothelial cells. (D) ChemR23 expression by wild-type but not ChemR23−/− murine neutrophils. (E) Permeabilized human neutrophils were stained with anti-ChemR23 (with goat-anti-mouse Alexa 488 secondary) and wheat germ agglutinin (WGA-Alexa 647) to visualize the cell membrane. Cells were analysed by confocal microscopy. (F) Human neutrophils were stained for ChemR23 and markers of secretory vesicles (CD35), specific granules (CD66b) and azurophil granules (CD63). (G) Neutrophils were loaded with Fura2-AM and calcium flux responses elicited by control (ionomycin), vehicle, ChemR23 inhibitor (CCX2005, 100 nM), C15 (10 pM), scrambled C15 (C15-S; 10 pM) or chemerin (1 nM). Responses are displayed as delta F340/F380. Graphs show mean values±s.e.m. from three to six independent experiments. *P<0.05 relative to vehicle-treated or pre-flow cells. alpha MSH, alpha-melanocyte-stimulating hormone; IL-8, interleukin-8; TNF alpha, tumour necrosis factor alpha. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/23999103), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of ChemR23/CMKLR1 by Immunocytochemistry/ Immunofluorescence

Non-genomic modulation of ChemR23 expression on neutrophils (A,B) ChemR23 expression on human leukocytes was determined by flow cytometry on vehicle and TNF alpha (10 ng/ml; 20 min)-stimulated cells. Representative histograms shown in A. (C) Neutrophils were treated with vehicle, pro-inflammatory (TNF alpha ; fMLF, 1 μM; IL-8, 100 ng/ml) or anti-inflammatory mediators (annexin A1, 10 nM; alpha MSH, 10 nM; C15, 10 pM) followed by staining for ChemR23. ChemR23 expression was also assessed on isolated human neutrophils before and after flow over activated endothelial cells. (D) ChemR23 expression by wild-type but not ChemR23−/− murine neutrophils. (E) Permeabilized human neutrophils were stained with anti-ChemR23 (with goat-anti-mouse Alexa 488 secondary) and wheat germ agglutinin (WGA-Alexa 647) to visualize the cell membrane. Cells were analysed by confocal microscopy. (F) Human neutrophils were stained for ChemR23 and markers of secretory vesicles (CD35), specific granules (CD66b) and azurophil granules (CD63). (G) Neutrophils were loaded with Fura2-AM and calcium flux responses elicited by control (ionomycin), vehicle, ChemR23 inhibitor (CCX2005, 100 nM), C15 (10 pM), scrambled C15 (C15-S; 10 pM) or chemerin (1 nM). Responses are displayed as delta F340/F380. Graphs show mean values±s.e.m. from three to six independent experiments. *P<0.05 relative to vehicle-treated or pre-flow cells. alpha MSH, alpha-melanocyte-stimulating hormone; IL-8, interleukin-8; TNF alpha, tumour necrosis factor alpha. Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/23999103), licensed under a CC-BY license. Not internally tested by R&D Systems.

Detection of ChemR23/CMKLR1 by Flow Cytometry

Competition binding assays on BMDCs.[A, B] FACS analysis showing the cell surface expression of mChemR23/Dez on BMDCs generated from wild-type or ChemR23−/− mice. Cells were incubated with an anti-mChemR23 antibody (open histogram) or a control isotype (filled histogram). [C, D] Competition binding assays were performed on BMDCs generated from wild-type (C) or ChemR23−/− mice (D). Purified cells were incubated with 0.2 nM 125I-CXCL12 as tracer, and CXCL12 (300 nM), chemerin (300 nM) or a monoclonal anti-CXCR4 antibody (10 µg/ml) as competitors. After one hour incubation, unbound tracer was separated by filtration and filters washed twice before counting. The data were normalized for non-specific binding (0%) and specific binding in the absence of competitor (100%). Statistical significance as compared to the 100% values was tested by two-way analysis of variance followed by Tukey's test (***, P<0.001; **, P<0.01). All data points were performed in triplicate and the displayed data are the mean of five experiments performed with three independent cell preparations (error bars indicate S.E.M.). Image collected and cropped by CiteAb from the following open publication (https://pubmed.ncbi.nlm.nih.gov/23469143), licensed under a CC-BY license. Not internally tested by R&D Systems.