The following antibodies (Abs) and other materials were used: phycoerythrin (PE)-conjugated anti-human HLA-A, B, C (W6/32, BioLegend), PE-conjugated anti-human MICA (159227, R&D Systems), PE-conjugated anti-CD107a (H4A3, SouthernBiotech), PE-conjugated anti-human IgG (polyclonal, Jackson ImmunoResearch), FITC-conjugated anti-KIR2DL2 (CH-L, BD Biosciences), FITC-conjugated anti-human IgG (polyclonal, Jackson ImmunoResearch), Alexa Fluor 647-conjugated streptavidin (Jackson ImmunoResearch), allophycocyanin (APC)-conjugated anti-mouse IgG (polyclonal, Jackson ImmunoResearch), Pacific Blue-conjugated anti-human CD16, purified/biotinylated anti-SUDV-GP (3C10), purified anti-B7H6, Capture: purified anti-human IFN-γ (NIB42, BioLegend), Detection: biotin anti-human IFN-γ (4S.B3, BioLegend), 7-aminoactinomycin D (7AAD) (BioLegend), anti-phosphotyrosine 4G10, anti-PLCγ1 (Upstate), anti-SHP-1 (Santa Cruz), anti-GAPDH (Biodesign), and p-nitrophenyl phosphate (pNPP; New England BioLabs). The following fusion-Igs were used as previously described; NKG2D-Ig (37 (link)), NKp30-Ig (38 (link)), NKp44-Ig (39 (link)), NKp46-Ig (40 (link)), KIR2DL4-Ig (41 (link)), KIR2DL1-Ig (27 (link)), and KIR2DL2-Ig (42 (link)).
Fitc conjugated anti human igg
Manufactured by Jackson ImmunoResearch
Sourced in United States
The FITC-conjugated anti-human IgG is a laboratory product that detects and binds to human immunoglobulin G (IgG) molecules. The FITC (fluorescein isothiocyanate) label allows for the visualization of the bound IgG through fluorescence microscopy or flow cytometry techniques.
Automatically generated - may contain errors
Lab products found in correlation
7 aminoactinomycin d 7 aad, by BioLegend (1 mentions)
Alexa fluor 647 conjugated streptavidin, by Jackson ImmunoResearch (1 mentions)
Anti shp1, by Santa Cruz Biotechnology (1 mentions)
Pe conjugated anti human igg, by Jackson ImmunoResearch (1 mentions)
P nitrophenyl phosphate pnpp, by New England Biolabs (1 mentions)
Calcium and magnesium, by Thermo Fisher Scientific (1 mentions)
Plant lectins, by Vector Laboratories (1 mentions)
Alexa fluor 488 conjugated streptavidin, by Thermo Fisher Scientific (1 mentions)
Human igg1 isotype control, by Enzo Life Sciences (1 mentions)
Anti flag antibody, by Merck Group (1 mentions)
Cytomics fc 500 mpl, by Beckman Coulter (1 mentions)
Mouse igg1 isotype control, by BD (1 mentions)
Four well chamber slide, by Thermo Fisher Scientific (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluorescence microscopy, by Keyence (1 mentions)
4 protocols using fitc conjugated anti human igg
1
Characterization of NK Cell Receptor Interactions
2
Lectin and Chimeric Receptor Binding Assay
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All the staining with plant lectins (Vector Laboratories) or Chimeric receptors (R&D System) were performed in 1% BSA in HBSS supplemented with calcium and magnesium (Gibco). For the analysis using plant lectins or in-house Fc chimeric proteins, 100.000 cells were incubated with 1 µg/mL of biotinylated plant lectin or 1 µg/mL Fc chimeras for 45 min at 4 °C. Siglec-Fc chimeras were pre-incubated FITC-conjugated anti-human IgG (Jackson ImmunoResearch) for 15 min at room temperature before adding to the cells. The detection of plant lectins was performed by incubating the cells with AlexaFluor 488-conjugated Streptavidin (Invitrogen).
When indicated, cells were treated with 30mU/mL of Neuraminidase from Arthrobacter ureafaciens for 30 min at 37 °C to study the sialic acid dependency of the interaction. To evaluate whether sialylated structures are present in N-glycans, O-glycans or Glycolipids, cells were treated for 3 days with 2 mM Benzyl-GalNAc, 10 µg/mL Kifunensine or 1 mM PPMP, respectively.
When indicated, cells were treated with 30mU/mL of Neuraminidase from Arthrobacter ureafaciens for 30 min at 37 °C to study the sialic acid dependency of the interaction. To evaluate whether sialylated structures are present in N-glycans, O-glycans or Glycolipids, cells were treated for 3 days with 2 mM Benzyl-GalNAc, 10 µg/mL Kifunensine or 1 mM PPMP, respectively.
3
Cell Surface Expression of B7-H3 Domains
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Full‐length human B7‐H3 (NCBI Reference Sequence, NP_001019907.1: a.a. 27–534), and IgV1 (a.a. 27–139), IgC1 (a.a. 140–244), IgV2 (a.a. 245–357), or IgC2 (a.a. 358–456) domain expression vectors, each with transmembrane/intracellular domain (a.a. 457–534), were transfected into CHO‐K1 cells. Each vector had FLAG‐tag at the N‐terminus. They were then treated with 1 μg/mL DS‐5573a or human IgG1 isotype control (Enzo Life Sciences, New York, NY, USA), and were stained with FITC‐conjugated anti‐human IgG (Jackson ImmunoResearch, West Grove, PA, USA). To detect the expressed B7‐H3 proteins on the cell surface, cells were treated with 1 μg/mL anti‐FLAG antibody (Sigma‐Aldrich, Tokyo, Japan) or mouse IgG1 isotype control (BD, Tokyo, Japan), and were stained with FITC‐conjugated anti‐mouse IgG (Cappel, Aurora, OH, USA). Samples were analyzed by Cytomics FC500 MPL (Beckman Coulter, Tokyo, Japan).
4
Immunofluorescent Staining of IgG
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For immunofluorescent staining, the cells were cultured on fourwell chamber slides (Thermo Fisher Scientific, Rockford, IL) or type I collagen-coated eight-well chamber slides (Becton, Dickinson and Company, Franklin Lakes, NJ). The cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 15 min. After permeabilization with 0.25% TritonX-100 for 10 min, the cells were incubated with 100-fold diluted FITC-conjugated anti-human IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) or anti-mouse IgG (Jackson ImmunoResearch, West Grove, PA). The nuclei were stained with DAPI (Life Technologies, Tokyo, Japan). Photoimages were taken with a Keyence fluorescence microscopy (Keyence, Osaka, Japan). IgG was quantified by ImageJ (http://rsbweb.nih.gov/ij/).
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