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38 | Chapter 2 and only a small subset represents CD56+CD16- regulatory cells 14 . Their function is tightly regulated by a delicate balance between inhibitory and activating receptors, among which CD16, a lowaffinity receptor for the Fc fragment of IgG 1 , enablesNK cell mediated cytotoxicity of IgG 1 -coated cells, a phenomenon known as antibody dependent cellular cytotoxicity (ADCC) 15 . Although NK cells are involved in the outcome of important clinical interventions that are frequently monitored by multi-colour flow cytometry, such as transplantation 16-18 or immunotherapy 19 , the existing multi-colour FACS panels for NK cell analysis are either restricted to detect antigens associated with malignant transformation 12 or if they include an extended immunophenotyping panel, their standardized implementation is limited by the fact that measurements have not been validated through harmonized procedures across multiple centers 20 . In this article, we describe the design and harmonization of two eight-colour NK FACS panels, allowing the generation of reproducible similar data sets across multiple centers, highlighting the advantages of using cryopreserved PBMC for phenotypic and functional immune monitoring studies of NK cells 21,22 . Results NK FACS panel establishment based on backbone and drop-in concept To harmonize multicolour flow cytometry analysis for studying NK cell phenotype and function, three independent research centers using different flow cytometers equipped with compatible laser and detector/filter settings (Table 1) tested comparability and reproducibility of obtained data sets between centers. To this end, instrument set-up, sample preparation, acquisition and data analysis were performed independently using standardized protocols, which were commonly agreed on and followed in all three centers as described in the materials and methods section (Figure 1). Acquisition protocols were set up in each center using single stains, complete mixture of all antibody-fluorochrome combinations and fluorescence minus one (FMO) controls (Supplementary Fig. S1 online). Compensation matrices of the NK phenotype and function panels were generated for the three flow cytometers and these settings were used through the whole study (Supplementary Tables S1, S2, and S3 online). FACS panels were designed using a backbone and drop-in concept. Antigens of the backbone specifically discriminated the viable NK cell subset from PBMC, leaving four other detectors available to characterize the specific NK cell phenotype or function. The backbone of the NK phenotype panel (summarized in Table 2) comprises a combination of CD3, TCRγδ, CD14 and CD19 lineage-specific antibodies (for negative selection of the NK cells), and CD45 and CD56 (for positive selection). In this respect it is important to remark on the extensive overlap between NK cells and γδT cells in terms of the