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56 | Chapter 2 sterical hindrance. For staining, the PBMC concentration was adjusted in washing buffer (PBS containing 0.5% bovine serum albumin) to 1 x 10 6 /ml and stained in 96 well U bottom plates (Corning, Amsterdam, The Netherlands). PBMC were incubated with relevant antibody mixes in a final premix volume of 30µl for 10 minutes at 4°C in the dark. After incubation cells were washed once with 170 μl of washing buffer per well and centrifuged at 300xg for 5min, supernatants were discarded, and cell pellets suspended in 200µl of washing buffer until measurement. Samples were measured within 1 hour. The antibody mix for the NK phenotype panel consisted of CD45 VioGreen, CD3 VioBlue, TCRγδ VioBlue, CD14 VioBlue, CD19 VioBlue and SYTOX ® Blue for live NK cell discrimination and NK cell phenotypic antigens PanKIR2D FITC, NKG2A PE, NKG2C PE-Vio770 and NKG2D PerCP-Cy5.5. Similarly, the antibody mix for the NK function panel consisted of CD45 VioGreen, CD14 VioBlue, CD19 VioBlue and SYTOX ® Blue, together with CD3 PerCP-Vio700 and TCRγδ PerCP-Vio700 to identify NK cells, with inclusion of NK function antigens CD25 VioBrightFITC, CD107a PE, NKp44 PE-Vio770. Most of the antibodies were supplied by Miltenyi Biotec except NKG2D PerCP-Cy5.5 (Biolegend, Fell, Germany) and dead cell stain SYTOX ® Blue (Thermo Fisher Scientific, Berlin, Germany). Manufacturer recommended dilutions of the antibodies were first checked and finally used. Details on antibody clones and dilutions used in the NK phenotyping and NK function panel are listed in tables 2 and 3. Due to the absence or low expression of some antigens (CD25, NKp44 or CD107a) on NK cells in unstimulated PBMC, we used Ig-capture beads (MACS ® Comp Bead anti-mouse Igκ and MACS ® Comp Bead anti-human Igκ, Miltenyi Biotec GmbH, Bergisch-Gladbach, Germany) to establish initial fluorescence compensation matrices using the automatic compensation feature of each instrument. Further, unstained PBMC and single staining of all antibodies was acquired to assess the compensation matrices obtained with the Ig- capture beads. In addition to this, FMO controls were performed for drop in antigens (CD25 VioBrightFITC, NKp44 PE-Vio770 and CD107a PE) in the NK function panel (Supplementary figure 1). Compensation matrices were calculated using automated settings from BD FACS DIVA TM software for BD FACSCanto TM II and BD LSRFortessa TM , and MACSQuantify TM 2.8 software for MACSQuant ® Analyzer X. In situations where instrument compensation was not optimal, adjustments were made based on the single staining controls from PBMC using KALUZA ® data analysis software (Beckman Coulter, California, US). Compensation matrices of the NK phenotype and function panels for the three flow cytometers are shown in supplementary tables S1, S2, and S3 online. NK cell cytotoxicity assays Activated and non-activated PBMC (containing effector cells) were stimulated with A431 cells (targets) in the presence or absence of 5μg/ml of cetuximab (CET) in a total volume of 100 µl in 96 well U bottom plates. PBMC counts were adjusted so that NK cell numbers