15353-j-veluchamy

2 Harmonized NK FACS panels to study NK cell phenotype and function | 55 in the NK cell phenotype and function between non-activated and cytokine activated samples, both fresh and cryopreserved samples were split into 2 fractions. One fraction was activated overnight with 1000U/ml of IL-2 (Proleukin®; Chiron, München, Germany) and 10ng/ml of IL-15 (CellGenix GmbH, Freiburg, Germany), while the other fraction was incubated without cytokines. Cell line The A431 (epidermoid carcinoma) cell line was obtained from ATCC and cultured in Dulbecco's modified medium (DMEM; Invitrogen, Carlsbad CA, USA) containing 100 U/ml penicillin, 100 µg/ml streptomycin and 10% fetal calf serum (FCS; Integro, Zaandam, The Netherlands). Cells were passaged every 5 days and early-passage cells (passage numbers between 20-25) were used for the experiments. Cells were maintained in a 37°C, 95% humidity, 5% CO 2 atmosphere. Anti - EGFR monoclonal antibody The anti-EGFR monoclonal antibody cetuximab (Erbitux ® , Merck, Darmstadt, Germany) was purchased from the VU University Medical Center pharmacy for NK cell ADCC experiments. Flow cytometers and instrument settings Experiments were performed with three different flow cytometers: BD LSRFortessa TM X-20 (Becton Dickinson (BD) B.V, Breda, The Netherlands), BD FACSCanto TM II (Becton Dickinson B.V, Breda, The Netherlands) and MACSQuant ® Analyzer X (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). All devices were equipped with 3 solid state lasers (Violet, Blue and Red). Comparable band pass and long pass filters enabled the use of same fluorochrome- conjugated antibodies between the three machines (Table 1). The chosen fluorochromes were distributed accordingly; two light scatter parameters and 8 fluorescence detectors: Violet laser excited VioGreen and VioBlue fluorochromes, Blue laser for forward and side scatter, besides detecting signals from FITC, VioBrightFITC, PE, PerCP-Cy5.5, PerCP-Vio700 and PE-Vio770 channels and red laser for APC and APC-Vio770. Daily maintenance and initial photomultiplier tube (PMT) voltage determination were performed using recommended cytometer tracking and set-up beads (CS&T) (BD biosciences, Breda, The Netherlands) for BD instruments, and MACSQuant ® calibration beads (Miltenyi Biotec GmbH, Bergisch- Gladbach, Germany) for MACSQuant ® Analyzer X. Antibodies, staining protocol and compensation settings The antibody clones and fluorochrome combinations were selected based on lab user experience and antibody sensitivity and with the aim of achieving minimal spill over between detectors. Moreover, the antibody - fluorochrome conjugates were chosen in order to attain high sensitivity for measuring dimly expressed antigens besides overcoming