mouse anti-human fcγriia clone iv.3  (STEMCELL Technologies Inc)

Journal: Rheumatology (Oxford, England)

Article Title: Immune complex-mediated neutrophil activation in patients with polymyalgia rheumatica

doi: 10.1093/rheumatology/keac722

Figure Lengend Snippet: FcγRIIA-mediated neutrophil activation induced by plasma from patients with PMR. Plasma from HCs ( n = 8) and patients with PMR ( n = 8) were incubated in vitro with neutrophils isolated from a healthy object in the presence or absence of the FcγRIIA-blocking antibody (Clone IV.3) for 120 min and assessed for its capacity to up-regulate the neutrophil activation surface makers CD66b ( A ) and CD11b ( B ). Results are shown as the mean fluorescence intensity (MFI). Statistical analyses were done using the Mann–Whitney U -test as well as the Wilcoxon signed-rank test; ** P < 0.01, *** P < 0.001. HC: healthy control; ns: not significant

Article Snippet: To assess neutrophil activation, neutrophils at a concentration of 3 × 10 5 cells/well were incubated in the presence or absence of the human FcγRIIA blocking antibody (IV.3) (5 μg/ml; Caprico Biotechnologies, Norcross, GA, USA) for 30 min before addition of stimuli, such as R848 (3.5 μg/ml), or plasma samples from patients with PMR ( n = 8) and HCs ( n = 8) (1:50 dilution) for an additional 120 min.

Techniques: Activation Assay, Clinical Proteomics, Incubation, In Vitro, Isolation, Blocking Assay, Fluorescence, MANN-WHITNEY, Control

Journal: PLoS ONE

Article Title: LyGDI, a Novel SHIP-Interacting Protein, Is a Negative Regulator of FcγR-Mediated Phagocytosis

doi: 10.1371/journal.pone.0021175

Figure Lengend Snippet: (A) THP-1 cells were stimulated for the indicated time points using human FcγRIIa/IIb (CD32) antibody followed by goat F(ab') 2 anti-mouse IgG. SHIP was immunoprecipitated from resting ‘R’ and activated ‘A’ cells and analyzed by Western blotting (IB) with anti-LyGDI (upper panel). Control IPs were performed using normal mouse IgG. The same membrane was reprobed with anti-phosphotyrosine (pY) (middle panel) and anti-SHIP (lower panel). Similar co-immunoprecipitation assays were performed from cells stimulated using (B) F(ab') 2 fragments of FcγRI antibody 32.2 and goat F(ab') 2 anti-mouse IgG secondary antibody, and (C) F(ab') 2 fragments of FcγRIIa antibody IV.3 and goat F(ab') 2 anti-mouse IgG secondary antibody.

Article Snippet: The F(ab') 2 of human FcγRI (32.2), human FcγRIIa (IV.3) and human FcγRIIIa (3 g8) antibody were obtained from Medarex (Princeton, NJ).

Techniques: Immunoprecipitation, Western Blot

Journal: PLoS ONE

Article Title: LyGDI, a Novel SHIP-Interacting Protein, Is a Negative Regulator of FcγR-Mediated Phagocytosis

doi: 10.1371/journal.pone.0021175

Figure Lengend Snippet: THP-1 cells were stimulated for different time points using anti-CD32 followed by goat anti-mouse F(ab') 2 IgG secondary antibody. (A) LyGDI was immunoprecipitated from resting and activated cells and analyzed by Western blotting with anti-Shc (upper panel). The same membrane was reprobed with anti-LyGDI to ensure equivalent protein loading (lower panel). Control IPs were done using normal goat IgG. (B) SHIP was immunoprecipitated from resting and activated cells and analyzed by Western blotting with anti-Shc (upper panel). The same membrane was reprobed with anti-SHIP to ensure equivalent protein loading (lower panel). Control IPs were done using normal mouse IgG. (C) SHIP was immunoprecipitated from resting and activated cells and analyzed by Western blotting with anti-Grb2 (upper panel). The same membrane was reprobed with anti-SHIP to ensure equivalent protein loading (lower panel). Control IPs were done using normal mouse IgG. (D) LyGDI was immunoprecipitated from resting and activated cells and analyzed by Western blotting with anti-Grb2 (upper panel). The same membrane was reprobed with anti-LyGDI to ensure equivalent protein loading (lower panel). Control IPs were done using normal goat IgG. All co-IP results are representative of at least three independent experiments.

Article Snippet: The F(ab') 2 of human FcγRI (32.2), human FcγRIIa (IV.3) and human FcγRIIIa (3 g8) antibody were obtained from Medarex (Princeton, NJ).

Techniques: Immunoprecipitation, Western Blot, Co-Immunoprecipitation Assay

Journal: PLoS ONE

Article Title: LyGDI, a Novel SHIP-Interacting Protein, Is a Negative Regulator of FcγR-Mediated Phagocytosis

doi: 10.1371/journal.pone.0021175

Figure Lengend Snippet: (A) 1×10 7 PBM were transfected with either scrambled or LyGDI siRNA. Western blotting was done to measure LyGDI after 48 hours (upper panel). The membrane was reprobed with anti-actin to ensure equivalent loading (lower panel). Numbers in the upper panel are mean optical density (arbitrary units) normalized to actin. (B) Control and LyGDI siRNA-transfected samples were tested for FcγR expression by flow cytometery analysis. Overlay histograms show FcγR staining in secondary-only antibody controls (gray), control siRNA-transfected (black) and si-LyGDI-transfected (red). (C) Control and LyGDI siRNA-transfected PBMs were stained with mouse anti-Rac followed by Alexa flour 555 conjugated to goat anti-mouse IgG, F(ab') 2 fragment. (D) The fold change in Rac membrane localization in control versus LyGDI siRNA-transfected PBMs was quantified as described in .

Article Snippet: The F(ab') 2 of human FcγRI (32.2), human FcγRIIa (IV.3) and human FcγRIIIa (3 g8) antibody were obtained from Medarex (Princeton, NJ).

Techniques: Transfection, Western Blot, Expressing, Staining

Journal: Cell reports

Article Title: Maternal Anti-Dengue IgG Fucosylation Predicts Susceptibility to Dengue Disease in Infants

doi: 10.1016/j.celrep.2020.107642

Figure Lengend Snippet:

Article Snippet: To assess FcγR expression cells were stained at 1:100 with a FITC conjugated anti-human FcγRIIa antibody, clone IV.3 (STEMCELL Technologies; 60012FI); Brilliant Violet 711 conjugated anti-human FcγRIII, clone 3G8 (Biolegend; 302044); an FcγRIIb antibody clone 2B6 (gifted by the Ravetch lab, Rockefeller University) conjugated to Alexa Fluor 647 (ThermoFisher; A20186) and an APC conjugated anti-human FcγRI, clone 10.1 (Biolegend, 305014) for 30 minutes at 4°C.

Techniques: Virus, Recombinant, Plasmid Preparation, Software