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108 | Chapter 4 streptavidin APC (BD biosciences, Netherlands). A nonspecific IgG 1 and IgG 2 specific APC labeled antibody was used as a negative control. DNA extraction and FCGR3AV158V and V158F (V/V and V/F) polymorphism genotyping Genomic DNA (gDNA) was isolated from MNCs using QIA amp DNA kit (QIAGEN, Westburg, The Netherlands). Purified DNA was eluted in a volume of 100µl. Purity and Quantity of gDNA was measured using the nanodrop method (NANODROP 1000, Thermo Scientific). About 40-200ng of gDNA was used for FcγRIIIa polymorphism assays. FcγRIIIa primers ID: C__42463377_10 were purchased from Life Technologies, The Netherlands. 40ng of gDNA, 6.25µl of PCR master mix and 12.5µl mix of forward and reverse primers with VIC and FAM labeled probes for V/V and V/F polymorphism respectively were added. Readings were interpreted using v2.0.1 software (Bio Rad, Netherlands). Known controls for VV and VF genotypes were included in the experiment. Flow cytometry Flow cytometry analysis was done on a BD LSRFORTESSA X-20 (BD Biosciences). Cell numbers and expression of cell-surface markers were determined by flow cytometry. The cell numbers and the population of live cells was determined by gating on CD45 + cells based on forward scatter (FSC) and side scatter (SSC). For analysis of phenotype, the cells were gated only on FSC/SSC and further analyzed for the specific antigen of interest. Cells were incubated with the appropriate concentration of antibodies for 30 min at 4°C. After washing, cells were suspended in FACS buffer. Flow cytometry-based cytotoxicity and degranulation studies Flow cytometry was used for the read-out of cytotoxicity assays. Target cells were labelled with 5µM pacific blue succinimidyl ester (PBSE; Molecular Probes Europe, Leiden, The Netherlands) in a concentration of 1x10 7 cells per ml for 10 min at 37°C. The reaction was terminated by adding an equal volume of FCS, followed by incubation at room temperature for 2 min after which stained cells were washed twice with 5 ml DMEM/10% FCS. After washing, cells were suspended in DMEM/10% FCS to a final concentration of 5 x 10 5 /ml. CD56 + NK cells were washed with PBS and suspended in Glycostem Basal Growth Medium (GBGM) + 2% FCS to a final concentration of 5 x 10 5 /ml. Target cells were co-cultured with effector cells at an E:T ratio of 1:1 in a total volume of 250 µl in 96-wells flat-bottom plates (5 x 10 4 targets in 100 µl of DMEM + 10% FCS incubated with 5 x 10 4 effectors in 100 µl of GBGM + 2% FCS, further supplemented with 25 µl of GBGM + 2% FCS and DMEM + 10% FCS medium). NK cells and target cells alone were plated out in triplicate as controls. Target cells were coated with anti-EGFR mAbs for 1h at 4°C. Cells (A431, COLO320, Caco-2,