Cd69 pe clone fn50

Antibodies including CD3-FITC (clone OKT3; 1:150, BioLegend, San Diego, CA, USA), CD56 PE/Cy7 (clone NCAM1; 1:50, BioLegend), CD69-PE (clone FN50; 1:60, BD Biosciences, Heidelberg, Germany), CD107a-PE (clone H4A3; 1:25, BD Biosciences) and the corresponding isotype controls were obtained from BioLegend. 7-AAD (BioLegend) staining (1:200) or LIVE/DEAD™ Fixable Violet (1:1000, Thermo Fisher Scientific, Waltham, MA, USA) were used to identify non-viable cells within flow cytometric samples. For flow cytometric assays, 30,000 target cells were incubated in 96-well plates with 65,000 PBMCs, Rituximab at 2.5 µg/mL and various tyrosine kinase inhibitors at the indicated concentrations. After 4 and 24 h, flow cytometric analyses were performed to determine CD107a and CD69 expression, respectively. NK cells were identified as CD56+ CD3+, activated subsets as CD69+. CD107a expression indicated degranulation. Detailed gating strategies are provided in Supplementary Figure S1. All samples were analyzed using the BD FACS Canto II or BD FACS Fortessa II (BD Biosciences, Heidelberg, Germany). Data analysis was performed using FlowJo V10 software (BD Bioscience).

Natural killer cytotoxicity and IFN-γ production were analyzed in co-cultures of primary NK cells and K562 cells (an NK-sensitive target cell line that lacks MHC-I molecules) with and without previous exposure of the NK cells to individual LRAs from our panel. Cytotoxicity was assessed by analyzing the expression of the degranulation marker CD107a, a reliable marker of NK cell cytotoxic activity (28 (link)). A total of 100,000 NK cells were co-cultured with the same number of K562 target cells in 96-well plates for 4–6 h in the presence of PE/Cy7-CD107a antibody, clone H4A3 (BD), adding 1 μL of GolgiStop (BD) after the first hour of culture. Cells were then harvested, washed, and surface-stained with CD56-FITC, clone NCAM 16 (BD) in staining buffer for 20 min on ice in the dark. Cells were then fixed with Fixation buffer (Biolegend, San Diego, CA, USA) during 20 min at room temperature in the dark, washed with Perm/Wash buffer twice and intracellularly stained with IFNγ-PE (Biolegend, San Diego, CA, USA) for 20 min. After washing, cells were re-suspended in staining buffer and analyzed in the Attune Focusing Cytometer (Applied Biosystems). To analyze whether NK cells were non-specifically activated by LRA, NK cells were also incubated in the absence of target cells, and CD69 (CD69-PE, clone FN50, from BD) and CD107a expression was analyzed as described.