Nkg2c

Manufactured by R&D Systems

NKG2C is a type II transmembrane glycoprotein expressed on the surface of natural killer (NK) cells. It functions as an activating receptor that recognizes and binds to specific ligands, triggering the activation of NK cells and their subsequent effector functions.

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Anti-NKG2C-PE (10 citations) NKG2C-PE (8 citations) NKG2C Alexa Fluor 700 (4 citations) PE-conjugated NKG2C (4 citations) NKG2C (134591) (3 citations) NKG2C-PerCP (3 citations) Anti-NKG2C (clone 134591) (2 citations) NKG2C-PE (clone 134591, (2 citations) Anti-NKG2C-PE (clone 134591) (2 citations) Anti-NKG2C-Alexa Fluor 488 (2 citations)

5 protocols using nkg2c

Flow Cytometry Analysis of Immune Cell Subsets

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For flow cytometry, fluorochrome-conjugated antibodies to the surface epitopes CD3 (OKT4; Biolegend), CD56 (HDCD56; Biolegend), CD57 (NK-1; BD Biosciences), NKG2C (134591; R&D Systems), and CD107a (H4A3; BD Biosciences) were used. Intracellular EAT-2 was stained using a rabbit polyclonal antibody (Proteintech Group) followed by secondary staining with fluorochrome-conjugated anti-rabbit reagents (Invitrogen). Intracellular staining of cytokines was performed using fluorochrome-conjugated antibodies against IFN-γ (B27; BD Biosciences) and TNF (MAb11; Biolegend). For phenotypic analyses, PBMCs or purified NK cells were surface stained with the indicated antibodies along with a fixable dead cell stain (Invitrogen) in FACS buffer (PBS supplemented with 2% FBS and 2 mM EDTA) and fixed in 2% formaldehyde. For intracellular staining, cells were permeabilized in 0.05% Triton X-100 (Sigma). Flow cytometry data were acquired on an LSR II instrument (BD Biosciences) and analyzed with FlowJo (v10, Tree Star). MitoTracker Deep Red, CellRox (ThermoFisher), and propidium iodide (Sigma) staining was performed according the manufacturer’s instructions. For sorting experiments, PBMCs were stained with the indicated antibodies and sorted to >95% purity on a FACS Aria II instrument (BD Biosciences).

Cichocki F., Wu C.Y., Zhang B., Felices M., Tesi B., Tuininga K., Dougherty P., Taras E., Hinderlie P., Blazar B.R., Bryceson Y.T, & Miller J.S. (2018). ARID5B regulates metabolic programming in human adaptive NK cells. The Journal of Experimental Medicine, 215(9), 2379-2395.

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Multiparametric Flow Cytometry Analysis

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Antibodies and clones used for phenotyping, intracellular staining, and cell sorting are listed in SI Appendix, Table S2. Secondary staining was performed with streptavidin Qdot 605 or Qdot 585 (both Invitrogen), anti-mouse IgM (II/41, eFluor 650NC; eBioscience), or streptavidin BB-630 or streptavidin-BV650 (BD Biosciences) and Live/Dead Aqua (Invitrogen). After surface staining, cells were fixed and permeabilized using a FoxP3/Transcription Factor staining kit (eBioscience) for subsequent intracellular staining. Purified NKG2C (no. 134591; R&D Systems) was biotinylated using a FluoReporter Mini-Biotin XX protein labeling kit (Life Technologies) and detected using streptavidin-Qdot 585, 605, or 655 (Invitrogen).
Samples were analyzed on a BD LSRFortessa equipped with four lasers (BD Biosciences) or a BD FACSymphony A5 equipped with five lasers (BD Biosciences), and data were analyzed using FlowJo version 9.5.2 and version 10.6.1 (Tree Star). UMAPs were constructed in FlowJo 10.6.1 using the UMAP plugin. UMAP coordinates and protein expression data were subsequently exported from FlowJo, and protein expression for each parameter was normalized to a value between 0 and 100. UMAP plots were made in R using ggplot, and color scale shows log2(normalized protein expression +1).

Brownlie D., Scharenberg M., Mold J.E., Hård J., Kekäläinen E., Buggert M., Nguyen S., Wilson J.N., Al-Ameri M., Ljunggren H.G., Marquardt N, & Michaëlsson J. (2021). Expansions of adaptive-like NK cells with a tissue-resident phenotype in human lung and blood. Proceedings of the National Academy of Sciences of the United States of America, 118(11), e2016580118.

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Comprehensive Immunophenotyping Protocol

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The following antibodies were used for flow cytometry: BD Bioscience: CD3 epsilon (UCHT1, SP34–2 and SK7), CD122 (Mik-β3), CD8a (RPA-T8), PLZF (RI7–809). CD107a (H4A3), IFN-γ (B27), KIR3DL1 (DX9), KIR2DL2/L3/S2 (CH-L), CD16 (3G8), CD57 (NK-1), HLA-DR (G46–6), HLA-C (DT9), and CD86 (FUN-1). Biolegend: CD2 (RPA-2.10), PD-1 (EH12.2H7), Bcl-2 (100), CD3 epsilon (SK7, OKT3, HIT3a and APA1/1), HLA-ABC (W6/32), NKp46 (9E2), IgG1 (MOPC-21), IgG2a (MOPC-173), CD5 (UCHT2), Lck (LCK-01), and Bcl11b (25E6). Beckman Coulter: CD56 (NKH-1), KIR2DL2/L3/S2 (GL183), KIR2DL1/S1 (EB6), NKp46 (BAB281), CD247 (TIA-2), CD69 (TP1.55.3). Miltenyi: NKG2A (REA110), NKG2C (REA205), PD-1(PD1.3.1.3), LAG-3 (REA351), CD58(TS2/9), Ki-67 (REA183), Bw4 (REA274), and TCRrd (11F2). R&D Systems: NKG2C (134591), KIR2DL1 (143211), and KIR2DL3 (180701). Millipore: FcεRI (FCABS400F), HCMV-IEA (8B1.2). ThemoFisher: LIR-1 (HP-F1), TCRab (WT13), and CD8b (SIDI8BEE). Abcam: CD3 gamma (EPR4517), CD3 delta (EP4426) and Bcl11b (25E6). Jackson Immunoresearch: Donkey anti-rabbit IgG (H+L). The following antibodies were used for western blot: Biolegend: CD3 epsilon (SK7), IgG1 (MOPC-21), β-actin (W16197A), CD247 (6B10.2), pLck (A18002D), Lck (LCK-01), CD2 (RPA-2.10), and CD16 (3G8); pCD247 (C415.9A, Santa Cruz Biotechnology), CD3 gamma (EPR4517, Abcam), and CD3 delta (EP4426, Abcam).

Wu Z., Lau C.M., Sottile R., Le Luduec J.B., Panjwani M.K., Conaty P.M., Srpan K., Laib Sampaio K., Mertens T., Adler S.P., Hill A.B., Barker J.N., Cheung N.K., Sun J.C, & Hsu K.C. (2021). Human Cytomegalovirus Infection Promotes Expansion of a Functionally Superior Cytoplasmic CD3+ NK Cell Subset with a Bcl11b-Regulated T Cell Signature. The Journal of Immunology Author Choice, 207(10), 2534-2544.

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Isolation and Phenotyping of Human NK Cells

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Peripheral blood was collected from healthy donors using protocols approved by the Memorial Sloan Kettering Cancer Center Institutional Review Board (nos. 06-107 and 95-054). Specific processing on these samples was performed under Biospecimen Research Protocol Institutional Review Board no. 16-1564. Donors provided informed written consent. PBMCs were isolated by Ficoll gradient purification. Flow cytometry and cell sorting were performed on the LSR II and Aria II cytometers (BD Biosciences), respectively. NK cells were sorted and/or phenotyped from PBMCs by using the following fluorophore-conjugated antibodies at a 1:50 dilution, unless otherwise indicated: DAPI, CD3ε (1:200 dilution, UCHT1, BD Biosciences), CD56 (1:100 dilution, N901, Beckman Coulter) and CD14 (M5E2, BD Biosciences), CD57 (HCD57, BioLegend) and NKG2C (134591, R&D). Cells were sorted to >95% purity.

Wiedemann G.M., Santosa E.K., Grassmann S., Sheppard S., Le Luduec J.B., Adams N.M., Dang C., Hsu K.C., Sun J.C, & Lau C.M. (2021). Deconvoluting global cytokine signaling networks in natural killer cells. Nature immunology, 22(5), 627-638.

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Comprehensive Immunophenotyping of NK Cells

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Thawed PBMC were stained with ethidium monoazide (EMA), anti-CD19-PeCy5, anti-CD3-AlexaFluor700 (both from BD Biosciences), and with either anti-CD14-V711 (Biolegend) or anti-CD14-PeCy5 (AbD Serotec, Raleigh, NC) to exclude dead cells, T cells, B cells and monocytes. NK cells were identified using anti-CD56-PeCy7 (BD Biosciences). FITC-conjugated antibodies against CD122, CXCR3 (R&D Systems), CD69 and HLA-DR (BD Biosciences), PE-conjugated antibodies against TRAIL, CD300 (BD Biosciences), NKG2A, NKG2D, and NKp44 (Beckman Coulter, Brea, CA), and APC-conjugated antibodies against CD85j (eBiosciences, San Diego, CA), CCR5 (BD Biosciences), NKG2C (R&D Systems), NKp30 and NKp46 (Miltenyi Biotec, Auburn, CA) were added. Liver-infiltrating lymphocytes were stained with anti-TRAIL-PE, anti-CD69-APC/Cy7 (BD Biosciences) and anti-NKp46-APC (Miltenyi Biotec) in addition to EMA and the lineage-specific antibodies described above.

Serti E., Chepa-Lotrea X., Kim Y.J., Keane M., Fryzek N., Liang T.J., Ghany M, & Rehermann B. (2015). Successful Interferon-Free Therapy of Chronic Hepatitis C Virus Infection Normalizes Natural Killer Cell Function. Gastroenterology, 149(1), 190-200.e2.

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