recombinant human l selectin igg1 fc chimera  (R&D Systems)

Journal: The Journal of investigative dermatology

Article Title: α(1,3) Fucosyltransferases IV and VII are essential for the initial recruitment of basophils in chronic allergic inflammation.

doi: 10.1038/jid.2013.160

Figure Lengend Snippet: Figure 4. Selectin-dependent cooperative recruitment of basophils and effector cells. (a) Irradiated a(1, 3) fucosyltransferase-IV (FT-IV)( / )/FT-VII( / ) mice received wild-type (WT) basophils in combination with CD49b( ) bone marrow cells (effector cells) from either WT or FT-IV( / )/FT-VII( / ) mice. They were then immunized with trinitrophenyl-IgE (TNP-IgE) and challenged with TNP-OVA. The untreated group comprised FT-IV( / )/FT-VII( / ) mice without cell transfer. (b) Cell populations in inflammatory skin. *Po0.05 compared with WT effector cells. BM, basement membrane.

Article Snippet: In vitro selectin binding assay of basophils CD49b (þ ) basophil–enriched bone marrow cells were suspended in phosphate-buffered saline containing 5% fetal calf serum, 0.1% NaN3, 1 mmol l 1 Ca2þ , and 1 mmol l 1 Mg2þ , followed by incubation with 10mg ml 1 of murine P-, E-, or L-selectin-human IgG Fc chimera or control human IgG1 Fc (R&D Systems) for 40 minutes at 4 1C.

Techniques: Irradiation, Membrane

Journal: The Journal of investigative dermatology

Article Title: α(1,3) Fucosyltransferases IV and VII are essential for the initial recruitment of basophils in chronic allergic inflammation.

doi: 10.1038/jid.2013.160

Figure Lengend Snippet: Figure 5. Expression of a(1, 3) fucosyltransferase (FT) messenger RNA (mRNA) and FT-dependent selectin binding in basophils. (a) Primary basophils were subjected to further purification by positive selection with CD123 (purity 499%). Transcripts for FT-IV and FT-VII mRNA were quantified by real-time PCR. (b) Binding of soluble P- and E-selectins to primary basophils assessed by flow cytometry. BMBa, bone marrow–derived basophil; BMMCs, bone marrow–derived mast cells.

Article Snippet: In vitro selectin binding assay of basophils CD49b (þ ) basophil–enriched bone marrow cells were suspended in phosphate-buffered saline containing 5% fetal calf serum, 0.1% NaN3, 1 mmol l 1 Ca2þ , and 1 mmol l 1 Mg2þ , followed by incubation with 10mg ml 1 of murine P-, E-, or L-selectin-human IgG Fc chimera or control human IgG1 Fc (R&D Systems) for 40 minutes at 4 1C.

Techniques: Expressing, Binding Assay, Selection, Real-time Polymerase Chain Reaction, Cytometry, Derivative Assay

Journal: The Journal of investigative dermatology

Article Title: α(1,3) Fucosyltransferases IV and VII are essential for the initial recruitment of basophils in chronic allergic inflammation.

doi: 10.1038/jid.2013.160

Figure Lengend Snippet: Figure 6. Basophil P-selectin glycoprotein-1 (PSGL-1)–L-selectin interaction involvement in IgE-mediated chronic allergic inflammation (IgE-CAI). (a) PSGL-1 and L-selectin expressions on basophils. (b) PSGL-1 Ab effect on selectin binding to primary basophils. (c) L-selectin binding to basophils from wild-type (WT) and a(1, 3) fucosyltransferase-IV (FT-IV)( / )/FT-VII( / ) mice. (d) Effects of L-selectin and/or PSGL-1 Abs on IgE-CAI in WT mice. *Po0.05 compared with control IgG. **Po0.05 compared with L-selectin or PSGL-1 Ab alone.

Article Snippet: In vitro selectin binding assay of basophils CD49b (þ ) basophil–enriched bone marrow cells were suspended in phosphate-buffered saline containing 5% fetal calf serum, 0.1% NaN3, 1 mmol l 1 Ca2þ , and 1 mmol l 1 Mg2þ , followed by incubation with 10mg ml 1 of murine P-, E-, or L-selectin-human IgG Fc chimera or control human IgG1 Fc (R&D Systems) for 40 minutes at 4 1C.

Techniques: Binding Assay, Control

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 1. SDS-PAGE (12.5% gel), showing purity of the SAP and soluble L-selectin preparations. In Western blots, SAP was equally detected by anti-SAP (lane 5) or L-selectin (lane 6) antibodies. From left to right: (1) Low-molecular-weight mar- kers, silver stained on blot; (2) SAP (100 ng/lane), silver stained on blot; (3) low-molecular-weight markers, silver stained in gel; (4) SAP (400 ng/lane), silver stained in gel; (5) SAP (800 ng/ lane), immunodetection with anti-SAP mAb CBL305; (6) SAP (1000 ng/lane), immunodetection with human L-selectin-IgG1 chimera in the presence of 5 mM Ca2+ followed by biotinylated mouse anti-human IgG antibody (Fc-specific); (7) soluble L- selectin (3000 ng/lane), silver stained in gel; (8) soluble L- selectin (1000 ng/lane), immunodetection with anti-human L- selectin mAb Dreg200.

Article Snippet: In a second series of experiments, wells were coated as above with human L-selectin-IgG1 Fc chimera or human IgG1 (both from R&D Systems).

Techniques: SDS Page, Western Blot, Molecular Weight, Staining, Immunodetection

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 2. SDS-PAGE (7.5% gel), showing purity of the human L- selectin-IgG1 chimera. In Western blots, the human L-selectin- IgG1 chimera was equally detected by anti-L-selectin antibody (lane 5), anti-human IgG Fc antibody (lane 6) or SAP (lane 7). From left to right: (1) High-molecular-weight markers, silver stained on blot; (2) human L-selectin-IgG1 chimera (100 ng/ lane), silver stained on blot; (3) high-molecular-weight mar- kers, silver stained in gel; (4) human L-selectin-IgG1 chimera (400 ng/lane), silver stained in gel; (5) human L-selectin-IgG1 chimera (13 ng/lane), immunodetection with biotinylated anti- human L-selectin mAb Dreg200; (6) human L-selectin-IgG1 chimera (25 ng/lane), immunodetection with biotinylated mouse anti-human IgG antibody (Fc-specific); (7) human L- selectin-IgG1 chimera (200 ng/lane), immunodetection with biotinylated SAP in the presence of 5 mM Ca2+.

Article Snippet: In a second series of experiments, wells were coated as above with human L-selectin-IgG1 Fc chimera or human IgG1 (both from R&D Systems).

Techniques: SDS Page, Western Blot, High Molecular Weight, Staining, Immunodetection

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 3. Binding of various concentrations of biotinylated SAP or anti-L-selectin mAb Dreg200 to L-, P- or E-selectin-IgG chimeras coated on microtiter wells. Bound SAP or antibody was detected luminometrically after reaction with ExtrAvidin- alkaline phosphatase (RLU, relative light units). Prior treatment of biotinylated SAP with sialidase or N-glycosidase did not result in altered binding curves. Virtually no binding of biotinylated SAP was observed to uncoated wells or to wells coated with any selectin chimera in the absence of Ca2+ (EDTA). Means SD of four independent experiments.

Article Snippet: In a second series of experiments, wells were coated as above with human L-selectin-IgG1 Fc chimera or human IgG1 (both from R&D Systems).

Techniques: Binding Assay

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 4. Impact of Dreg200 or Dreg55, murine IgG1 control, heparin, fucoidan, asialofetuin or a2-macroglobulin on the binding of biotinylated SAP (1 lg/mL in TBS containing 5 mM Ca2+) to wells coated with human L-selectin-IgG1 chimera. Means of three independent experiments (SD never exceeded 0.035 RLU).

Article Snippet: In a second series of experiments, wells were coated as above with human L-selectin-IgG1 Fc chimera or human IgG1 (both from R&D Systems).

Techniques: Control, Binding Assay

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 6. Ca2+-dependent interaction of SAP and soluble L- selectin purified from human plasma. Commercially obtained SAP was subjected to native 4–15% gradient PAGE after reaction with soluble L-selectin, Ca2+, or EDTA. Immunodetection of SAP with anti-SAP mAb CBL305 after blotting on polyvinylidene difluoride membrane. From left to right: (1) SAP (15 pmol monomer/lane) + L-selectin (3 pmol/lane) + 2.5 mM Ca2+; (2) SAP (15 pmol monomer/lane) + L-selectin (3 pmol/lane) + 2.5 mM EDTA; (3) SAP (7.5 pmol monomer/lane) + L-selectin (3 pmol/lane) + 2.5 mM Ca2+; (4) SAP (7.5 pmol monomer/lane) + L-selectin (3 pmol/lane) + 2.5 mM EDTA; (5) SAP (15 pmol monomer/lane) + 2.5 mM Ca2+; (6) SAP (7.5 pmol monomer/ lane) + 2.5 mM Ca2+.

Article Snippet: In a second series of experiments, wells were coated as above with human L-selectin-IgG1 Fc chimera or human IgG1 (both from R&D Systems).

Techniques: Purification, Clinical Proteomics, Immunodetection, Membrane

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 7. SPR measurements demonstrating Ca2+-dependent binding of mouse L-selectin-IgG*protein G*gold conjugates to immobilized SAP. Reaction of mouse L-selectin-human IgG*protein G*gold conjugates (20 nm or 40 nm diameter) to SAP covalently attached to a Biacore CM5 chip (track 2), as opposed to BSA (track 1). The four cycles show the difference in RU between track 2 and track 1 at a flow rate of 40 lL/min. The left arrow of each cycle marks the injection at the start of the cycle. Cycle 1: 20-nm gold conjugate + 1 mM Ca2+; cycle 2: 20- nm gold conjugate + 3 mM EDTA; cycle 3: 40-nm gold conjugate + 1 mM Ca2+; cycle 4: 40-nm gold conjugate + 3 mM EDTA. The right arrows of the four cycles mark the start points of injections of EDTA buffer without conjugate to regenerate the chip. X axis: time (minutes).

Article Snippet: In a second series of experiments, wells were coated as above with human L-selectin-IgG1 Fc chimera or human IgG1 (both from R&D Systems).

Techniques: Binding Assay, Injection

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 8. In flow chamber assays with an effective wall shear stress of 2.0–2.6 dyn/cm2 (representative for L-selectin-depen- dent adhesion), the number of adherent neutrophils per high power field (HPF) to confluent layers of TNF-a-stimulated (500 U/mL for 4 h at 37C; TEBU GmbH, Germany) or un- stimulated (control) human umbilical cord endothelial cells was counted with or without SAP (0.1 or 1 lmol/L) or the anti-L- selectin antibody Dreg200 (50 lg/mL) being present during the experiment (means SD; n = 3–9). The insert shows adhesion of neutrophils to TNF-a-activated endothelial cells in the presence of varying concentrations of Dreg 200 (black; 50, 10, or 1 lg/mL), murine IgG1 isotype (light grey; 50, 10, or 1 lg/mL) and fucoidan (dark grey; 50, 5, or 0.5 lg/mL).

Article Snippet: In a second series of experiments, wells were coated as above with human L-selectin-IgG1 Fc chimera or human IgG1 (both from R&D Systems).

Techniques: Shear, Control

Journal: European journal of immunology

Article Title: Binding of human serum amyloid P componentto L-selectin.

doi: 10.1002/eji.200425360

Figure Lengend Snippet: Figure 9. Up-regulation of the lymphocyte activation-depen- dent integrin neoepitope (mAb 24 epitope) by 10 min of incubation at 37C with 3 mM Mn2+ (positive control, top) or with 5 lg/mL SAP (bottom). Filled histograms: Staining at 0 min (identical to IgG1 negative control histograms at 0 and 10 min). Full-line histograms: Expression at 10 min. Dotted-line histo- gram: Partial inhibition by Dreg200 Fab.

Article Snippet: In a second series of experiments, wells were coated as above with human L-selectin-IgG1 Fc chimera or human IgG1 (both from R&D Systems).

Techniques: Activation Assay, Incubation, Positive Control, Staining, Negative Control, Expressing, Inhibition