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Nkp46 clone 29a1

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NKp46 (clone 29A1.4) is a monoclonal antibody that recognizes the NKp46 receptor, a key activating receptor expressed on natural killer (NK) cells. This receptor plays a crucial role in the function and cytotoxicity of NK cells, which are important effector cells of the innate immune system.

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Lab products found in correlation

Lsrii flow cytometer, by BD (2 mentions) Cd4 clone rm 4 5, by Thermo Fisher Scientific (2 mentions) 100 μm cell strainer, by Corning (2 mentions) Red blood cell lysing buffer hybri max, by Merck Group (2 mentions) Clone tonbo 2, by Thermo Fisher Scientific (2 mentions) H 2kb clone af6 88, by Thermo Fisher Scientific (2 mentions) H 2db clone 28 14 8, by Thermo Fisher Scientific (2 mentions) Anti mouse isotype igg2a clone ebm2a, by Thermo Fisher Scientific (2 mentions) Cd8α clone 53 6, by Thermo Fisher Scientific (2 mentions) Anti mouse cd45 clone 30 f11, by BioLegend (2 mentions) 35 μm cell strainer, by Corning (2 mentions) Facscalibur, by BD (1 mentions) Canto 2, by BD (1 mentions) Cd127 clone a7r34, by Thermo Fisher Scientific (1 mentions) Ultrapure formaldehyde, by Polysciences (1 mentions) Ifn γ clone xmg1, by Thermo Fisher Scientific (1 mentions) Cd45 clone 30 f11, by BD (1 mentions) Cd3 clone 145 2c11, by BD (1 mentions) Cd4 clone gk1, by BD (1 mentions) T bet clone ebio4b10, by Thermo Fisher Scientific (1 mentions)

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Anti-NKp46 (clone 29A1.4, (2 citations)

4 protocols using nkp46 clone 29a1

1

Multiparametric Flow Cytometry Analysis

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To perform surface staining, 1 × 106 cells were placed in individual wells of a 96-well round bottom plate and incubated with the appropriate antibody cocktails for 15 min at 4°C on a slow rocker. After the staining, cells were fixed in a solution of 2% ultrapure formaldehyde (Polysciences, Inc., Warrington, PA, USA) in FACS buffer for 20 min on ice, washed twice, and analyzed the following day on the Canto II (BD Biosciences) or FACSCalibur (BD Biosciences). Intracellular staining was performed using Cytofix/Cytoperm Fixation/Permeabilization Solution Kit with BD GolgiStop (BD biosciences) according to the manufacturer’s instruction. Flow cytometry acquisition was performed on an LSRIISorp. Data were analyzed using FACS Express or FlowJo software (Tree Star, Inc., Ashland, OR, USA). Antibodies against CD45 (clone 30-F11, BD Pharmingen), CD3 (clone 145-2C11, BD Pharmingen), CD4 (clone GK1.5, BD Pharmingen), CD8 (clone 5H10, Biolegend), T-bet (clone eBio4B10, eBioscience), IL-17A (clone ebio17B7, eBioscience), IL-4 (clone B11B, Biolegend), IFNγ (clone XMG 1.2, eBioscience), IL-22 (clone A3.6M, eBioscience and clone poly5164, Biolegend), TGF-β (clone 11A5, Biolegend), IL-17F (clone ebio18F10, eBioscience), NKP46 (clone 29A 1.4, Biolegend), c-Kit (clone 2B8, Biolegend), Sca-1 (clone D7, BD Pharmingen), and CD127 (clone A7R34, eBioscience) were used.
Shen W., Hixon J.A., McLean M.H., Li W.Q, & Durum S.K. (2016). IL-22-Expressing Murine Lymphocytes Display Plasticity and Pathogenicity in Reporter Mice. Frontiers in Immunology, 6, 662.
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2

Detecting Endogenous Gp100-Specific CD8+ T Cells

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For the detection of endogenous gp100-specific CD8+ T cells, we used an H-2Kb-KVPRNQDWL pentamer (ProImmune, Oxford, UK). Skin and draining LNs were analyzed for the presence of H-2Kb-KVPRNQDWL pentamer-positive CD8+ T cells. The cells were stained according to the manufacturer's protocol, and unspecific pentamer binding was excluded by pregating on viable CD19− (clone 1D3, BD Biosciences) NKp46− (clone 29A1.4, Biolegend) cells. For the detection of IFN-γ, we incubated cell suspensions from draining LNs with 1 μM gp100 peptide (KVPRNQDWL, Peptide&Elephants, Potsdam, Germany) for 48 h at 37 °C. IFN-γ release and antibody staining were performed as described above. For the calculation of gp100-responsive IFN-γ+ CD8+ cells, we used this formula: ((mean of IFN-γ+ CD8+ cells restimulated with gp100 peptide)−(mean of IFN-γ+ CD8+ cells cultured without gp100 peptide))/(mean of unstimulated pentamer+ CD8+ cells).
Mairhofer D.G., Ortner D., Tripp C.H., Schaffenrath S., Fleming V., Heger L., Komenda K., Reider D., Dudziak D., Chen S., Becker J.C., Flacher V, & Stoitzner P. (2015). Impaired gp100-Specific CD8+ T-Cell Responses in the Presence of Myeloid-Derived Suppressor Cells in a Spontaneous Mouse Melanoma Model. The Journal of Investigative Dermatology, 135(11), 2785-2793.
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3

Comprehensive Immune Cell Profiling

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The following anti-mouse antibodies were purchased from ThermoFisher and used according to manufacturer’s specifications: CD8α (clone 53–6.7, Cat#47–0081-30 or clone Tonbo 2.43, Cat# 50–1886-U100), CD4 (clone RM4–5, Cat #48–0042-82), H-2Kb (clone AF6–88.5.5.3, Cat #12–5958-82), H-2Db (clone 28–14-8, Cat #11–5999-82), anti-mouse isotype IgG2a (clone eBM2a, Cat# 114724–81). Anti-mouse CD45 (clone 30-F11, Cat# 103129) and NKp46 (clone 29A1.4, Cat# 137617) were purchased from Biolegend. Mouse spleens were passed through a 100 μm cell strainer (Corning Life Sciences) and incubated in Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich) for 3 min at room temperature. For detection of MHC-I, MOE/E6E7 cells were lifted with 1× citric saline to prevent cleavage of MHC-I on cell surface. The prepared cells were incubated with the corresponding panel of antibodies conjugated with unique fluorophores for 30 min to 1 h at room temperature and washed with PBS. Samples were passed through a 35 μm cell strainer (Corning Life Sciences) immediately before analysis on an LSRII flow cytometer (Becton Dickinson) using FACSDiva collection software.
Westrich J.A., Vermeer D.W., Silva A., Bonney S., Berger J.N., Cicchini L., Greer R.O., Song J.I., Raben D., Slansky J.E., Lee J.H., Spanos W.C, & Pyeon D. (2019). CXCL14 suppresses human papillomavirus-associated head and neck cancer through antigen specific CD8+ T cell responses by upregulating MHC-I expression. Oncogene, 38(46), 7166-7180.
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4

Comprehensive Immune Cell Profiling

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The following anti-mouse antibodies were purchased from ThermoFisher and used according to manufacturer’s specifications: CD8α (clone 53–6.7, Cat#47–0081-30 or clone Tonbo 2.43, Cat# 50–1886-U100), CD4 (clone RM4–5, Cat #48–0042-82), H-2Kb (clone AF6–88.5.5.3, Cat #12–5958-82), H-2Db (clone 28–14-8, Cat #11–5999-82), anti-mouse isotype IgG2a (clone eBM2a, Cat# 114724–81). Anti-mouse CD45 (clone 30-F11, Cat# 103129) and NKp46 (clone 29A1.4, Cat# 137617) were purchased from Biolegend. Mouse spleens were passed through a 100 μm cell strainer (Corning Life Sciences) and incubated in Red Blood Cell Lysing Buffer Hybri-Max (Sigma-Aldrich) for 3 min at room temperature. For detection of MHC-I, MOE/E6E7 cells were lifted with 1× citric saline to prevent cleavage of MHC-I on cell surface. The prepared cells were incubated with the corresponding panel of antibodies conjugated with unique fluorophores for 30 min to 1 h at room temperature and washed with PBS. Samples were passed through a 35 μm cell strainer (Corning Life Sciences) immediately before analysis on an LSRII flow cytometer (Becton Dickinson) using FACSDiva collection software.
Westrich J.A., Vermeer D.W., Silva A., Bonney S., Berger J.N., Cicchini L., Greer R.O., Song J.I., Raben D., Slansky J.E., Lee J.H., Spanos W.C, & Pyeon D. (2019). CXCL14 suppresses human papillomavirus-associated head and neck cancer through antigen specific CD8+ T cell responses by upregulating MHC-I expression. Oncogene, 38(46), 7166-7180.
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