Quantifying NK Cell DAP12 Expression

Images were taken using Leica AM TIRF MC imaging system as described with the following modifications [108 (link)]. NK-cells were isolated by negative selection and placed on glass chamber slides (5x10⁴ cells/chamber; LabTek II) precoated with 10μg/mL α-Ly49H mAb. Cells were stimulated for 15 minutes, fixed with 4% paraformaldehyde, and permeabilized with 0.25% Triton-X. Cells were blocked with SEA blocking buffer (Thermo-Fisher) for 1 hour and stained with 5 μL rabbit α-human/mouse DAP12 antibody (ab219765, Abcam) overnight at 4°C. Cells were washed and incubated with DyLight 488-conjugated donkey α-rabbit IgG (poly4064, BioLegend) secondary antibody for 2 hrs at room temperature. Cells were washed and fresh PBS was added to each well. Images were taken at room temperature using 100X oil submersion lens and Leica AM TIRF MC imaging system at the University of Iowa Central Microscopy Research Facility. Laser intensity and exposure parameters remained constant within each experiment. TIRF microscopy images were analyzed using ImageJ software. Membrane DAP12 was quantified by measuring mean pixel intensity in the longest axis of cells.