Anti nkp46 29a1

Manufactured by BioLegend

Anti-NKp46 (29A1.4) is a mouse monoclonal antibody that recognizes the NKp46 receptor expressed on natural killer (NK) cells. NKp46 is a major activating receptor involved in the recognition and lysis of certain tumor and virus-infected cells by NK cells. The antibody can be used for the identification and analysis of NK cells by flow cytometry.

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5 protocols using anti nkp46 29a1

Multiparametric Flow Cytometry Analysis

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The harvested spleens were mashed and single-cell suspensions of the spleens were stained with the following surface antibodies: anti-CD45 (30-F11, BD Biosciences), LIVE/DEAD Fixable Blue cell stain (Invitrogen), anti-CD19 (ID3, BD Biosciences), anti-CD8a (53-6.7, BioLegend), anti-CD27 (LG7F9, eBioscience), anti-CD11b (M1/70, BioLegend), anti-CD11c (HL3, BD Biosciences), anti-CD86 (GL1, BioLegend), anti-NK1.1 (PK136, BioLegend), anti-NKp46 (29A1.4, BioLegend), anti-MHC Class II (M5/114.15.2, BD Biosciences), anti-F4/80 (BM8, BioLegend), anti-CD80 (16-10A1), anti-CD3 (17A2, BioLegend), anti-CD4 (RM4-5, Invitrogen), anti-CTLA-4 (UC10-4B9, BioLegend), anti-PD-1 (29F.1A12, eBioscience), anti-CD28 (37.51, BioLegend), anti-CD44 (IM7, BD Biosciences), anti-CD43 (1B11, BioLegend), anti-CD47 (miap301, BioLegend), anti-CD62L (MEL-14, BioLegend), anti-CD25 (PC61.5, eBioscience), and anti-CD107a (1D4B, BioLegend). Intracellular staining was then performed with a FOXP3 permeabilization and fixation kit (eBioscience) according to manufacturer’s recommendations using the following antibodies: Ki-67 (B56; BD Biosciences) and GrB (QA16A02, BioLegend). Flow cytometric data were collected with a Beckman Coulter Cytoflex LX (6-L NUV) flow cytometer and analyzed using FlowJo software (Tree Star).

Rao D., O’Donnell K.L., Carmody A., Weissman I.L., Hasenkrug K.J, & Marzi A. (2021). CD47 expression attenuates Ebola virus-induced immunopathology in mice. Antiviral research, 197, 105226.

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Multiparametric Flow Cytometry Analysis

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Cell surface marker expression was analyzed on a Becton Dickinson LSR Fortessa^TM flow cytometer after staining with fluorochrome-conjugated antibodies. Intracellular staining was performed using the fix/perm buffer set (Biolegend), according to the manufacturer's instructions. Antibodies used were conjugated anti-NKp30 (P30–15, Biolegend), anti-NKp44 (P44–8.1, BD), anti-NKp46 (29A1.4, Biolegend), anti-IFN-γ (485.B3, Biolegend), anti-TNF-α (MAb11, eBioscience), anti-RANKL (MIH24, Biolegend), anti-Ki-67 (16A8, Biolegend), anti-CD107a (H4A3, BD), anti-CD57 (HCD57, Biolegend), anti-CD56 (N901, Beckman Coulter), anti-CD14 (HCD14, Biolegend), anti-CD16 (3G8, Biolegend), and anti-CD3 (UCHT1, Biolegend). Annexin V/7-AAD staining was performed according to the manufacturer's protocol (Biolegend). The data were analyzed using FlowJo v10.2 software.

Cribbs A., Hookway E.S., Wells G., Lindow M., Obad S., Oerum H., Prinjha R.K., Athanasou N., Sowman A., Philpott M., Penn H., Soderstrom K., Feldmann M, & Oppermann U. (2018). Inhibition of histone H3K27 demethylases selectively modulates inflammatory phenotypes of natural killer cells. The Journal of Biological Chemistry, 293(7), 2422-2437.

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Multiparameter Flow Cytometry of Immune Cells

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Isolated cells from thymic glands, PDLNs, and spleens were stained with the following antibodies: anti‐CD11b (M1/70; BioLegend), anti‐CD11c (N418; BioLegend), anti‐PDCA‐1 (129c1; BioLegend), anti‐Ly6G (1A8; BioLegend), anti‐CD8ɑ (53‐6.7; BD BioSciences), anti‐B220 (RA3‐6B2; BioLegend), anti‐CD19 (1D3; BD BioSciences), anti‐CD19 (6D5; BioLegend), anti‐CD5 (53‐7.3; BD BioSciences), anti‐CD21 (7E9; BioLegend), anti‐CD3 (17A2; BioLegend), anti‐NK1.1 (PK136; BioLegend), anti‐NKp46 (29A1.4; BioLegend), and anti‐IFN‐γ (XMG1.2; BioLegend). Dilutions, catalogue numbers, and RRIDs of antibodies are given in Table S1. The stained cells were analyzed by a LSR Fortessa at the BioVis core facility, Uppsala University, Uppsala, Sweden. Single stained, unstained, and Fluorescence Minus One controls were applied as negative controls to identify any spread of the fluorochromes into the channel of interest and to properly gate the fluorochromes. The data were analyzed by Flowlogic software (Inivai Technologies). Gating strategies for flow cytometric analysis for different cell types are shown in Figures S2‐S6.

Luo Z., Soläng C., Mejia‐Cordova M., Thorvaldson L., Blixt M., Sandler S, & Singh K. (2019). Kinetics of immune cell responses in the multiple low‐dose streptozotocin mouse model of type 1 diabetes. FASEB bioAdvances, 1(9), 538-549.

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Comprehensive NK Cell Phenotyping

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The following antibodies were purchased from eBioscience: anti-NK1.1 (PK136), anti-CD45.1 (A20), anti-CD45.2 (104), anti-H-2D^b (28-14-8), anti-CD107a (1D4B), anti-IFN-γ (XMG1.2), anti-NKG2D (MI-6, (24)), anti-H-2K^b (AF6-88.5), and anti-CD107a (1D4B), anti-FoxP3 (150D/E4), and anti-CD226 (10E5). From Biolegend we purchased anti-CD3-ε (145-2C11), and anti-NKp46 (29A1.4). From R&D systems we purchased anti-pan-RAE-1 (199215).
Biotin-conjugated mAbs were detected with streptavidin Pacific Blue (Invitrogen) or streptavidin Brilliant Violet 421 (BioLegend). Before staining, cells were preincubated for 20 min with 2.4G2 hybridoma supernatant to block FcγRII/III receptors. Flow cytometry was performed on a cytometer (LSR II, LSR Fortessa, or LSR Fortessa X-20, BD Biosciences), and data were analyzed with the FlowJo software (Tree Star, Inc.).
Where applicable, donor cells were gated based on the expression of a congenic CD45 molecule. NK cells were defined as CD3⁻NK1.1⁺ or CD3⁻NKp46⁺ cells.

Shifrin N.T., Kissiov D.U., Ardolino M., Joncker N.T, & Raulet D.H. (2016). Differential role of hematopoietic and non-hematopoietic cell types in the regulation of NK cell tolerance and responsiveness. Journal of immunology (Baltimore, Md. : 1950), 197(10), 4127-4136.

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Depleting NK cells and blocking NKp46 in mice

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To deplete NK cells, B6 mice were treated i.p. with 200 μg of anti-NK1.1 (PK136, Bio X Cell). To block NKp46 mice were treated i.p. with 50 μg non-depleting LEAF purified anti-NKp46 (29A1.4, Biolegend) (Narni-Mancinelli et al., 2012 (link)). Antibody treatments were started 5 weeks after infection with ME49 and continued every other day for 2 weeks for flow cytometry assays or until non-treated animal groups died from reactivation of T. gondii. No treatment animals were treated with 200 μl of 1 X PBS i.p.

Ivanova D.L., Krempels R., Denton S.L., Fettel K.D., Saltz G.M., Rach D., Fatima R., Mundhenke T., Materi J., Dunay I.R, & Gigley J.P. (2020). NK Cells Negatively Regulate CD8 T Cells to Promote Immune Exhaustion and Chronic Toxoplasma gondii Infection. Frontiers in Cellular and Infection Microbiology, 10, 313.

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