Anti cd69 bv421 clone fn50

Streptavidin Dynabeads were coated with biotinylated PVR (CD155), Nectin-2 (CD112), biotin, anti-KIR2DL1, anti-KIR2DL3, anti-KIR2DL5 and anti-KIR3DL1 as described before. 2.5 x 10⁴ cells of each reporter cell line were seeded into a well of a tissue culture-treated 96-well plate and co-incubated with 10 μl of the protein-coated beads for 5 h at 37°C/5% CO₂ in a final volume of 200 μl. For blocking experiments, prior to co-incubation with beads, KIR2DL5⁺ JRC were blocked for 30 min with 30 μg/ml purified anti-KIR2DL5 or 30 μg/ml purified IgG1 isotype control antibody. Blocking antibodies remained in the wells during the whole assay. After co-incubation, cells were washed with PBS and stained with the viability dye LIVE/DEAD Fixable Near-IR (Life Technologies), anti-CD3-BUV395 (clone UCHT1, BD Biosciences), anti-CD69-BV421 (clone FN50, Biolegend) and the appropriate KIR antibody conjugated to PE (anti-KIR2DL1-PE (clone REA284), anti-KIR2DL3-PE (clone REA147), anti-KIR2DL5-PE (clone UP-R1), anti-KIR3DL1-PE (clone DX9) (Miltenyi). Cells were fixed in CellFix (BD Biosciences) and CD69 expression as a readout for KIR crosslinking was analyzed by flow cytometry.

C1R cells expressing comparable levels of HLA-A2 D227K/T228A, wild-type HLA-A2, or HLA-A2 A245V/K^b were pulsed for 1 h with various concentrations of the indicated peptides. Cells were then washed twice with RPMI 1640 medium containing 100 U/mL penicillin and 100 μg/mL streptomycin and resuspended in R10. Each assay included 1.5 × 10⁵ peptide-pulsed C1R cells and 5 × 10⁴ MEL5 TCR⁺ CD8⁺ J.RT3-T3.5 cells. Unpulsed targets were used as negative controls. Expression of CD69 on the surface of MEL5 TCR⁺ CD8⁺ J.RT3-T3.5 cells was measured after 6 h using the following directly conjugated monoclonal antibodies: anti-CD8-α–PE-Cy7 (clone RPA-T8; Thermo Fisher Scientific), anti-CD8-β–eFluor660 (clone SIDI8BEE; Thermo Fisher Scientific), anti-CD69–BV421 (clone FN50; BioLegend), and anti-HLA-A2–FITC (clone BB7.2; BioLegend). Nonviable cells were excluded from the analysis using LIVE/DEAD Fixable Aqua (Thermo Fisher Scientific). Data were acquired using a NovoCyte Flow Cytometer (ACEA Biosciences) and analyzed using FlowJo software version 10.6.1 (FlowJo LLC).