CD107a degranulation assay and detection of phosphorylated proteins by flow cytometry

NK92 and YTS cell lines were assessed for surface CD107a expression as a marker for degranulation after activation. 2 × 10⁵ WT or CD56-KO NK92 and YTS cell lines were added to 12-well flat-bottomed plates that were pre-coated overnight at 4°C with 5 µg/ml anti-NKp30 (clone P30-15, Biolegend) and -CD18 (clone IB4) or 5% bovine serum albumin as a negative control. Alternatively, YTS cells were cocultured with 10⁵ 721.221 target cells at a 2:1 effector to target ratio in 5 ml polystyrene round-bottom tubes. Each condition was run in triplicate. Anti-CD107a antibody (eBioscience, clone H4A3) was added at the onset of incubation for co-culture experiments. Cells added to the pre-coated antibody plates were spun briefly at 200 rpm before incubation for 90 min at 37°C 5% CO₂. Cells activated by plate-bound antibodies were mechanically dislodged and transferred to 5 ml polystyrene round-bottom tubes and incubated for an additional 25 min with anti-CD107a. Cells were fixed using 300–400 µl of 2% paraformaldehyde (Electron Microscopy Sciences), then CD107a surface expression was measured using a modified LSR Fortessa (BD Biosciences) with the CD107a⁺ gate defined by unstained negative control cells. Data were analyzed using FlowJo 10 (BD Biosciences). For experiments performed with target cell activation the average background of media alone was subtracted from samples.

For detection of phospho-Pyk2, cells were activated on plates as above and then fixed and permeabilized with BD Cytofix/Cytoperm (BD Biosciences) followed by detection with pPyk2 Y402 (Abcam, ab4800, 1:50) and secondary detection with goat anti-rabbit IgG Alexa Fluor 488 (Invitrogen, 1:100). Mean fluorescence intensity was adjusted to relative fluorescent intensity by normalizing to the WT condition.