Kir2dl1 fc

Manufactured by R&D Systems

KIR2DL1-Fc is a recombinant protein that consists of the extracellular domain of the human KIR2DL1 (Killer Cell Immunoglobulin-Like Receptor 2DL1) fused to the Fc region of human IgG1. KIR2DL1 is an inhibitory receptor expressed on natural killer (NK) cells that recognizes certain HLA-C allotypes. The Fc region provides a means for detection and purification of the fusion protein.

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Lab products found in correlation

Kir2dl2 fc, by R&D Systems (2 mentions) Streptavidin dynabeads, by Thermo Fisher Scientific (1 mentions) Tigit fc, by R&D Systems (1 mentions) F ab 2 goat anti human igg pe secondary antibody, by Thermo Fisher Scientific (1 mentions) Lsrfortessa, by BD (1 mentions) Bio plex 200, by Bio-Rad (1 mentions) Luminex xmap technology, by Bio-Rad (1 mentions) Ctb fitc, by Merck Group (1 mentions) Amaxa nucleofactor kit, by Lonza (1 mentions) Macs separation column, by Miltenyi Biotec (1 mentions)

Close spelling variants of this product

KIR2DL1

3 protocols using kir2dl1 fc

Quantifying Protein-Ligand Interactions

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Streptavidin Dynabeads (Thermo Fisher Scientific) were coated with either 200 pmol biotinylated protein or 10 μg biotinylated IgG/mg Dynabeads. Ligand-screening was performed by coating with biotinylated CD155 (PVR) (AcroBiosystems), CD112 (Nectin-2) (BPSBiosciences) or biotin as a negative control and anti-KIR2DL5 as a positive control. To screen for interactions with HLA class I and class II molecules, the LABScreen Single Antigen Class I and II kits (OneLambda) were used. Negative control beads were not coated with HLA antigens and positive control beads were coated with purified human IgG. Recombinant human Fc constructs (KIR2DL1-Fc, KIR2DL3-Fc, KIR2DL4-Fc, KIR2DL5-Fc, KIR3DL1-Fc, CD96-Fc, DNAM-1-Fc, LAG-3-Fc, PVR(CD155)-Fc, TIGIT-Fc) (R&D Systems) were diluted to 250 μg/ml in PBS and co-incubated with coated beads for 15 min at 4°C at a final concentration of 25 μg/ml. Samples were washed and bead-bound Fc constructs were detected by a staining with F(ab)2 goat-anti-human IgG PE secondary antibody (Life Technologies) for 15 min at 4°C. Interactions between Fc construct and peptide-coated beads were either quantified by flow cytometry (LSR Fortessa (BD Biosciences)) or by using the Luminex xMAP technology on a Bio-Plex 200 (Bio-Rad Laboratories).

Fittje P., Hœlzemer A., Garcia-Beltran W.F., Vollmers S., Niehrs A., Hagemann K., Martrus G., Körner C., Kirchhoff F., Sauter D, & Altfeld M. (2022). HIV-1 Nef-mediated downregulation of CD155 results in viral restriction by KIR2DL5+ NK cells. PLoS Pathogens, 18(6), e1010572.

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Flow Cytometry Staining of Fusion Proteins

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Fusion proteins LIR-1 Fc (kindly provided by Ofer Mandelboim), KIR2DL1-Fc and KIR2DL2-Fc (R&D Biosystems) were reconstituted in PBS (100μg/mL) and stored at −80°C. Cholera toxin B subunit (CTB)-FITC (Sigma) was reconstituted in sterile water (500μg/mL) and used at 10 μg/mL. Trypsinized cells were washed in PBS with 3% FCS or 0.5% BSA and stained in 40μL (fusion) protein dilution for 30min to 2h on ice. For fusion proteins, cells were washed twice and incubated on ice in 40μl secondary antibody APC AffiniPure F(ab’)₂ Fragment Goat Anti-Human IgG, Fcγ fragment specific (Jackson ImmunoResearch) (KIR2DL1, KIR2DL2) or mouse anti-human IgG (MH161–1, Sanquin) in-house conjugated to DL650 (Thermo Fisher Scientific) for 30–45min. After two washes cells were resuspended in PBS/3%FCS containing DAPI and analyzed on BD flow cytometers.

Jongsma M.L., de Waard A.A., Raaben M., Zhang T., Cabukusta B., Platzer R., Blomen V.A., Xagara A., Verkerk T., Bliss S., Kong X., Gerke C., Janssen L., Stickel E., Holst S., Plomp R., Mulder A., Ferrone S., Claas F.H., Heemskerk M.H., Griffioen M., Halenius A., Overkleeft H., Huppa J.B., Wuhrer M., Brummelkamp T.R., Neefjes J, & Spaapen R.M. (2020). The SPPL3-Defined Glycosphingolipid Repertoire Orchestrates HLA Class I-Mediated Immune Responses. Immunity, 54(1), 132-150.e9.

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Reconstituting MHC-I Expression in 721.221 Cells

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The class-1 MHC-negative human B cell line 721.221 (transduced with the TAP inhibitor ICP47) [24 (link)] was transfected with HLA-B*53:01, HLA-B*27:05, HLA-C*05.01, HLA-C*06.02, HLA-C*07.01, or HLA-C*08:02 (kindly provided by Prof. J. Strominger, Harvard University, USA) using the Amaxa nucleofactor kit (Lonza). Surface expression of HLA molecules on 721.221-ICP47-transfected cells was analyzed by flow cytometry using the pan–anti-HLA class-1 monoclonal antibody (#W6/32, Thermo-Fisher Scientific) as previously described [25 (link)].
NK-cell lines were obtained from 3 characterized healthy donors: NK-NAM: 2DL1⁻2DL2⁺2DL3⁻3DL1⁺; NK-DEP: 2DL1⁻2DL2⁻2DL3⁺3DL1⁺, and NK-RIM: 2DL1⁺2DL2⁻2DL3⁺3DL1⁻ [26 (link)]. NK cells were purified by using magnetic-activated cell sorting (MACS) separation columns (Miltenyi Biotec) and cultured in the presence of 100 U /mL recombinant IL-2 (Proleukin-2; Prometheus), as previously described [27 (link)]. NK cells were analyzed with KIR2DL1-Fc, KIR2DL2-Fc, or KIR3DL1-Fc fusion proteins (R&D Systems) followed by a secondary staining with a goat anti-human Fc-specific fragment-phycoerythrin antibody (Thermo-Fisher Scientific), as described [21 ].

Wauquier N., Petitdemange C., Tarantino N., Maucourant C., Coomber M., Lungay V., Bangura J., Debré P, & Vieillard V. (2019). HLA-C-restricted viral epitopes are associated with an escape mechanism from KIR2DL2+ NK cells in Lassa virus infection. EBioMedicine, 40, 605-613.

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