Nkp46 bv421

Manufactured by BioLegend

NKp46-BV421 is a fluorochrome-conjugated antibody that specifically binds to the NKp46 receptor, which is expressed on the surface of natural killer (NK) cells. It can be used for the identification and enumeration of NK cells in flow cytometry applications.

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5 protocols using nkp46 bv421

Dendritic cell subsets isolation

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Dendritic cell-enriched cell suspensions were stained with an antibody cocktail consisting of the following antibodies in PBS + 2% human sera for 30 min on ice: CD1c-APC/Cy7 (L161; BioLegend), CD3-BUV395 (UCHT1; BD Bioscience), CD11b-A700 (M1/70; BioLegend), CD11c-PE/Cy7 (3.9; BioLegend), CD14-A700 (HCD14; BioLegend), CD19-V450 (HIB19; BD Bioscience), CD20-eFluor 450 (2H7; eBioscience), CD56-BV421 (5.1H11; BioLegend), CD123–BV605 (6H6; BioLegend), CD141-BV711 (1A4; BD Bioscience), CD303-PerCP/Cy5.5 (201A; BioLegend), HLA-DR-PE-CF594 (G46-6; BD Bioscience), and NKp46-BV421 (9E2; BioLegend). After washing the cells, they were resuspended in PBS + 2% human sera + 0.1 mg/ml 4′,6-diamidino-2-phenylindole and cell sorted using a BD FACSAria II cell sorter into CD1c⁺ DCs (CD3⁻CD11b⁻CD14⁻CD19⁻CD20⁻CD56⁻CD123⁻NKp46⁻HLA–DR⁺CD11c⁺CD1c⁺), CD141⁺ DCs (CD1c⁻CD3⁻CD11b⁻CD14⁻CD19⁻CD20⁻CD56⁻CD123⁻NKp46⁻HLA-DR⁺CD11c⁺CD141⁺), pDCs (CD1c⁻CD3⁻CD11b⁻CD14⁻CD19⁻CD20⁻CD56⁻NKp46⁻HLA-DR⁺CD123⁺CD303⁺), and monocytes (CD3⁻CD19⁻CD20⁻CD56⁻CD123⁻NKp46⁻HLA-DR⁺CD11b⁺CD14⁺). The purity of sorted cell populations was reanalyzed and routinely above 95%.

Heger L., Balk S., Lühr J.J., Heidkamp G.F., Lehmann C.H., Hatscher L., Purbojo A., Hartmann A., Garcia-Martin F., Nishimura S.I., Cesnjevar R., Nimmerjahn F, & Dudziak D. (2018). CLEC10A Is a Specific Marker for Human CD1c+ Dendritic Cells and Enhances Their Toll-Like Receptor 7/8-Induced Cytokine Secretion. Frontiers in Immunology, 9, 744.

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Multiparametric Analysis of CAR-NK Cells

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All samples were stained with anti-CD56-APC, anti-CD3-FITC (Biolegend) and 7AAD (BD Biosciences). Transgene expression was detected by flow cytometry on 7AAD⁻ CD56(-APC)⁺ CD3(-PE)⁻ cells (Biolegend). For NK-cell receptor detection, samples were stained with DAPI, CD56-BV711, CD16-BV786, NKp30-AF647, NKp44-PE, NKp46-BV421 (Biolegend), NKG2D-APC (BD Biosciences), and NKG2A-PE (Miltenyi Biotec). CD3-BV650 and CD19-APC-Cy7 (Biolegend) markers were used as a gating exclusion strategy for the NK cell staining. Receptor expression was assessed on DAPI⁻ CD56(-BV711)⁺ CD3(-BV650)⁻ cells. To detect CAR-expression, cells were incubated with 2 μl Siglec2(CD22)-Fc chimera (50 mg/ml, R&D) for 30 min at 4°C, washed and stained with anti-Fc-PE (Jackson Immune).

Colamartino A.B., Lemieux W., Bifsha P., Nicoletti S., Chakravarti N., Sanz J., Roméro H., Selleri S., Béland K., Guiot M., Tremblay-Laganière C., Dicaire R., Barreiro L., Lee D.A., Verhoeyen E, & Haddad E. (2019). Efficient and Robust NK-Cell Transduction With Baboon Envelope Pseudotyped Lentivector. Frontiers in Immunology, 10, 2873.

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NK Cell Activation by Dendritic Cells

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NK cells were enriched from PBMCs by the depletion of unwanted cells using biotinylated antibodies and MoJo Streptavidin Nanobeads (BioLegend); more details are available in SI Appendix. The enriched NK cells were stained with CD3-BV605 (clone: UCHT-1, BioLegend), CD19-BV605 (clone: SJ25C1, BioLegend), CD20-BV605 (clone: 2H7, BioLegend), HLA-DR-BV510 (clone: L243, BioLegend), CD56-BV421 (clone: 5.1H11, BioLegend) and NKp46-BV421 (clone: 9E2, BioLegend) and sorted as CD56⁺NKp46⁺CD3⁻CD19⁻CD20⁻HLA-DR⁻ cells. Sorted XCR1⁻ and XCR1⁺ cDC1 and NK cells were cocultured in a 1:5 ratio and either stimulated with a cocktail of pIC (2.5 µg/mL), R848 (2.5 µg/mL), and CpG (2.5 µg/mL) or cultured in DC-medium alone. For some experiments, DCs and NK cells were separated in Transwell plates (96 well, 0.4 µm insert). NK cells were cultured in the well, while DCs were added to insert. In order to analyze the influence of secreted cytokines on NK cells, NK cells were cultured alone and supernatants of stimulated DCs were added corresponding to a 1:5 DC:NK cell ratio. After 18 h, cells were harvested and analyzed by flow cytometry for activation of NK cells (CD69). The supernatants were stored at −80 °C until analysis with LEGENDplex Human Interferon Panel (BioLegend) for secretion of IFNγ.

Heger L., Hatscher L., Liang C., Lehmann C.H., Amon L., Lühr J.J., Kaszubowski T., Nzirorera R., Schaft N., Dörrie J., Irrgang P., Tenbusch M., Kunz M., Socher E., Autenrieth S.E., Purbojo A., Sirbu H., Hartmann A., Alexiou C., Cesnjevar R, & Dudziak D. (2023). XCR1 expression distinguishes human conventional dendritic cell type 1 with full effector functions from their immediate precursors. Proceedings of the National Academy of Sciences of the United States of America, 120(33), e2300343120.

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Comprehensive Murine NK Cell Immunophenotyping

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The following antibodies were purchased from Biolegend: CD3 BV510, CD49b (DX5) APC-Cy7, NKp46 BV421, CD11b PE, Ly49D PE, Streptavidin BV650, CD27 biotin, KLRG1 BV605, KLRG1 PerCP/Cy5.5, CD69 PerCP/Cy5.5, and Ki67 APC. The following antibodies were purchased from eBioscience: CD107a PerCP-eFluor710, Ly49I PE, CD94 FITC, IFNγ APC, and Ly49H PE-Cyanine7. The following antibodies were purchased from BD Biosciences: NKG2A/C/E BV605. The following antibodies were purchased from Miltenyl Biotec: NKG2D (CD314) biotin, Ly49D biotin, and Ly49D PerCP-Vio700. To determine viability Invitrogen Fixable live/dead Aqua stain was used.

Ivanova D.L., Fatima R, & Gigley J.P. (2016). Comparative Analysis of Conventional Natural Killer Cell Responses to Acute Infection with Toxoplasma gondii Strains of Different Virulence. Frontiers in Immunology, 7, 347.

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Innate IEL Isolation and Characterization

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Cells were first incubated with anti-mouse CD16/CD32 Ab (clone 2.4 G2, BD Biosciences) for 10 min at 4° C, then washed and labeled with a cocktail of antibodies for 20 min at 4°C in dark (see Table 2). Cells were washed and treated with BD FACS Lysing Solution (BD Biosciences) for 5 minutes at RT. After washing, cells were analyzed on the LSR Fortessa X20 cytometer (Becton Dickinson).
For cell sorting, innate IELs were labeled with a cocktail of antibodies (CD45 FITC (Sony), CD103 PeCy7 (Biolegend) and NKp46 BV421 (Biolegend). Four populations (CD45⁺, CD45⁺NKp46^-, CD45⁺CD103^-NKp46^-, and CD45⁺CD103⁺) were sorted using a BD FACSAria II SORP cell sorter (Becton Dickinson).

Hariss F., Delbeke M., Guyot K., Zarnitzky P., Ezzedine M., Certad G, & Meresse B. (2023). Cytotoxic innate intraepithelial lymphocytes control early stages of Cryptosporidium infection. Frontiers in Immunology, 14, 1229406.

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