mouse anti human dcir alexa fluor 405  (R&D Systems)

Journal: JCI Insight

Article Title: Agonistic anti-DCIR antibody inhibits ITAM-mediated inflammatory signaling and promotes immune resolution

doi: 10.1172/jci.insight.176064

Figure Lengend Snippet: ( A ) Human monocytes were treated with 5 μg/mL antibodies for 30 minutes. Antibody binding was quantitated by the 10 μg/mL PE anti-human IgG secondary antibody. Data are normalized to the isotype group. Representative data from 2 independent studies are shown. ( B ) Scheme of the immunosuppressive effect induced by the agonistic anti-DCIR mAb. ( C ) Human monocytes were treated with 5 μg/mL anti-DCIR mAbs or isotype for 30 minutes, followed by immunoprecipitation (IP) using anti-SHP2 antibody. SHP2-DCIR interaction was evaluated by the DCIR level analyzed by WB. SHP2 from IP lysate and GAPDH from whole cell lysate were probed as loading controls. ( D and E ) Human monocytes were pretreated with 5 μg/mL antibodies for 30 minutes, followed by 50 μg/mL anti-human IC: HSA stimulation for 30 minutes. SYK’s interactions with SHP2 and FcRγ chain were evaluated by immunoprecipitation using anti-SHP2 ( D ) and anti-FcRγ ( E ) antibodies. SHP2 and FcRγ chain from IP lysate and Actin from whole cell lysate were probed as loading controls. ( A and C – E ) Representative data from 2 studies are shown. ( F and G ) Human monocytes ( n = 3) were pretreated with 10 μg/mL antibodies for 30 minutes, followed by 25 μg/mL ZymD stimulation overnight. Induction of TNF-α and IL-6 in supernatant were measured by ELISA and normalized to the isotype group. ( H ) Human neutrophils were pretreated with 10 μg/mL antibodies for 2 hours, and OCR were detected in real time after GM-CSF/PMA stimulation. AUC of the OCR was shown. Representative data from 3 independent studies are shown. ( F – H ) Means ± SEM are shown, and statistical significance is determined by 1-way ANOVA test with Dunnett’s correction compared with the isotype condition. * P < 0.05, ** P < 0.01.

Article Snippet: Tissue sections were prepared from paraffin blocks and stained with H&E, anti-DCIR antibody (MilliporeSigma, HPA007842), and rabbit IgG isotype control (MilliporeSigma, SAB5500149).

Techniques: Binding Assay, Immunoprecipitation, Enzyme-linked Immunosorbent Assay

Journal: JCI Insight

Article Title: Agonistic anti-DCIR antibody inhibits ITAM-mediated inflammatory signaling and promotes immune resolution

doi: 10.1172/jci.insight.176064

Figure Lengend Snippet: ( A ) LPS primed human neutrophils were labeled CellTracker Green. Neutrophils were incubated with 5 μg/mL anti-DCIR mAbs (3A4, 9D9, 5E11, 3F7) or isotype for 30 minutes on ice. Neutrophils and monocyte-derived macrophages were cocultured (ratio 4:1). ( B ) ADCP were quantitated by flow cytometry detecting CellTracker Green + CD11b + CD66b – HLA-DR + macrophage after 2 hours coculture with 3A4 ( n = 12), 9D9 ( n = 6), 5E11 ( n = 6), 3F7( n = 12), or isotype ( n = 11). Data are normalized to 3A4 group. ( C ) LPS primed neutrophils were incubated with 3A4, 9D9, 5E11, 3F7, or isotype for 30 minutes on ice. Neutrophils and Jurkat cells containing an ADCC reporter were cocultured (ratio 4:1). ( D ) ADCC were quantitated by luminescence overnight. Representative data from 2 studies are shown. ( E ) Confocal flow cytometry of human monocytes treated with 10 μg/mL A647-labeled antibodies (red) for 0, 15, and 30 minutes at 37°C. Antibody internalization was determined by colocalization (yellow) of A647-labeled antibodies in lysosome, marked by A488 anti-LAMP1 antibody (green). ( F and G ) HuDCIR-KI mice received i.p. administration of 10 mpk 5E11 with WT ( n = 25) or LALA mutant ( n = 7) huIgG1 Fc or isotype ( n = 25) 1 day before the i.p. injection of 0.5 mL 2 mg/mL ZymD. Relative leukocytes and neutrophils induction in peritoneal lavage were quantitated 6 hours after the ZymD injection. Data are normalized to isotype group. ( H – K ) Relative leukocytes and neutrophils infiltration ( H and I ) and cytokine in peritoneal fluid ( J and K ) collected 6 hours after ZymD injection, from mice pretreated with 5E11 with mouse IgG2b (5E11-mIgG2b) ( n = 5), Fc-matched isotype (iso-mIgG2b) ( n = 5), or anti–Gr-1 mAb (RB6-8C5) ( n = 5) 1 day before the peritonitis. Data are normalized to isotype group. Each dot represents 1 biological replicate. Means ± SEM are shown, and statistical significance is determined by 1-way ANOVA with Dunnett’s correction compared with the isotype condition. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Tissue sections were prepared from paraffin blocks and stained with H&E, anti-DCIR antibody (MilliporeSigma, HPA007842), and rabbit IgG isotype control (MilliporeSigma, SAB5500149).

Techniques: Labeling, Incubation, Derivative Assay, Flow Cytometry, Mutagenesis, Injection