Kir2dl2 fc

Manufactured by R&D Systems

KIR2DL2-Fc is a soluble recombinant protein that consists of the extracellular domain of the human Killer Cell Immunoglobulin-like Receptor 2DL2 (KIR2DL2) fused to the Fc region of human IgG1. KIR2DL2 is an inhibitory receptor expressed on natural killer (NK) cells and a subset of T cells.

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Lab products found in correlation

Kir2dl3 fc chimera, by R&D Systems (2 mentions) Protein a alexa fluor 488, by Thermo Fisher Scientific (2 mentions) Kir2dl1 fc, by R&D Systems (2 mentions) Ctb fitc, by Merck Group (1 mentions) Amaxa nucleofactor kit, by Lonza (1 mentions) Macs separation column, by Miltenyi Biotec (1 mentions)

4 protocols using kir2dl2 fc

KIR-Fc Chimera Binding Assay

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KIR2D2L2‐IgG and KIR2DL3‐IgG fusion constructs (KIR2DL2‐Fc & KIR2DL3‐Fc Chimera; R&D Systems) were conjugated with protein A Alexa Fluor 488 (Invitrogen) at a molar ratio of 6:1. A total of 1 × 10⁶ 721.174 cells were incubated with 10 μM peptide as described above and then stained with KIR‐Fc. After fixation in 4% w/v paraformaldehyde cells were analyzed by flow cytometry.

Cassidy S., Mukherjee S., Myint T.M., Mbiribindi B., North H., Traherne J., Mulder A., Claas F.H., Purbhoo M.A., Das J, & Khakoo S.I. (2014). Peptide selectivity discriminates NK cells from KIR2DL2‐ and KIR2DL3‐positive individuals. European Journal of Immunology, 45(2), 492-500.

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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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KIR2DL2-Fc and KIR2DL3-Fc Binding Assay

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KIR2D2L2-IgG and KIR2DL3-IgG fusion constructs (KIR2DL2-Fc & KIR2DL3-Fc Chimera; R&D Systems) were conjugated with protein A Alexa Fluor 488 (Invitrogen) at a molar ratio of 6:1. 1 × 10⁶ 721.174 cells were incubated with 10 μM peptide as described above and then stained with KIR-Fc. After fixation in 4% (w/v) paraformaldehyde cells were analyzed by flow cytometry.

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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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