To analyze the responsiveness of splenic NK cells ex vivo, 96-well high-binding flat-bottom plates (ThermoFisher (Waltham, MA)) were coated overnight with the following antibodies: control IgG 10 μg/mL, BioXCell); anti-NKG2D (clone CX5, BioLegend, San Diego, CA) 5 μg/mL; and anti-NKp46 (clone 29A1.4, BioLegend) 5 μg/mL. Plates then washed three times with PBS prior to stimulation. Single cell suspensions were generated from recipient splenic tissue of mice that had undergone transplantation, depleted of erythrocytes with red blood cell lysis buffer (Sigma-Aldrich), and cultured in the coated plates for five hours in the presence of monensin (2 uM) (Invitrogen (Carlsbad, CA)) and fluorophore (FITC)-conjugated anti-CD107a (0.5 g/mL) (Clone 1D4B, BioLegend). After stimulation, cells were stained with CD3 and NK1.1 to identify NK cells, followed by flow cytometric analysis.
Anti nkp46 clone 29a1
Manufactured by BioLegend
Anti-NKp46 (clone 29A1.4) is a monoclonal antibody that recognizes the NKp46 (also known as NCR1) receptor expressed on natural killer (NK) cells. NKp46 is a key activating receptor involved in the regulation of NK cell cytotoxicity and cytokine production.
Automatically generated - may contain errors
Lab products found in correlation
Monensin, by Thermo Fisher Scientific (1 mentions)
Red blood cell lysis buffer, by Merck Group (1 mentions)
Control igg, by BioXCell (1 mentions)
96 well high binding flat bottom plates, by Thermo Fisher Scientific (1 mentions)
Anti nk1.1 clone pk136, by BioLegend (1 mentions)
Anti cd49b clone dx5, by BioLegend (1 mentions)
Facsaria iiiu cell sorter, by BD (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Anti cd3 clone 17a2, by BioLegend (1 mentions)
Facsdiva, by BD (1 mentions)
2 protocols using anti nkp46 clone 29a1
1
Analyzing splenic NK cell responsiveness ex vivo
2
Cell Line Transfection and Flow Cytometry
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NIH3T3, CHO-K1, HEK 293T, and RMA cells were grown in IMDM, Ham’s F12, and RPMI medium, respectively, supplemented with 10% fetal calf serum (FCS) and 2 mM L-glutamine and transfected using the Lipofectamine 2000 reagent (Life Technologies), following the manufacturer’s instructions. The NIH3T3 and CHO-K1 transfectants were maintained in 0.5 mg/ml geneticin, and SCT Dd or SCT Dd-CD4 cell surface expression was regularly assessed by flow cytometry (clones 5-85 and 34-2-12, Abcam and Biolegend, respectively). The transfectants were FACS-sorted (FACSDiva and FACSARIAIIIU cell sorter, BD) for the cell surface expression of the relevant molecule, and stable transfectants were further selected using 0.8 mg/ml geneticin. Protein cell surface expression was detected by flow cytometry (FACSDiva, BD) using anti-NKG2D (clone C7, Biolegend), anti-Ly49A (clone YE1/48.10.6, Biolegend), anti-CD3 (clone 17A2, Biolegend), anti-NK1.1 (clone PK136, Biolegend), anti-NKp46 (clone 29A1.4, Biolegend), anti-CD49b (clone DX5, Biolegend), and anti-H60a (clone 205326, R\&D systems) antibodies.
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