ADCC assay for intracellular natural killer (NK) cell interferon-gamma (IFNγ) and CD107a expression was conducted as previously described, and analyzed with the same gating strategy [10] (link). Briefly, 96-well plates were coated overnight at 4 °C with chimeric cH9/1 HA protein (1 μg/ml). The next day, plates were washed with PBS and incubated with heat-inactivated sera (sera dilution 1:10) for two hours at 37 °C. Plates were washed again with PBS and incubated with 105 CD16 176v NK-92 cells per well (mycoplasma-free, human NK cell line expressing high affinity 176 V variant CD16 receptor) (Fox Chase Cancer Center, Philadelphia, PA, USA). As a negative control, NK-92 cells lacking expression of CD16 were added to an additional well for each sample.
The cells were incubated with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD) for 16 h at 37 °C. After incubation, cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen), anti-CD3-PE CF594 (BD) and anti-CD56-APC (BD) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend). Cells were acquired on BD Fortessa. Data analysis was done using FlowJo version 10 (treeStar).
The cells were incubated with anti-CD107a-AF488 antibody (Biolegend, San Diego, CA, USA), Brefeldin A (5 μg/ml, BD) and monensin (5 μg/ml, BD) for 16 h at 37 °C. After incubation, cells were stained with LIVE/DEAD Fixable Aqua dead cell staining kit (Invitrogen), anti-CD3-PE CF594 (BD) and anti-CD56-APC (BD) before intracellular staining with anti-IFN-γ-BV-421 (Biolegend). Cells were acquired on BD Fortessa. Data analysis was done using FlowJo version 10 (treeStar).
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