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2 Harmonized NK FACS panels to study NK cell phenotype and function | 39 expression of several NK cell receptors 23 ; therefore, we included an anti-TCRγδ antibody in the lineage cocktail to avoid misinterpretation of results . Viable CD45 + CD56 + NK cells were gated and CD16 APC, PanKIR2D FITC, NKG2A PE-Vio770, NKG2C PE and NKG2D PerCP-Cy5.5 (drop ins) were plotted against CD56 to quantify the percentage of NK cells positive for activating (CD16, NKG2C and NKG2D) and inhibitory (KIR2D and NKG2A) receptors (Table 2 and Supplementary Fig. S2 online). The backbone of the NK cell function panel, was slightly modified to acquire simultaneous information on NK cell, T cell and CD3+CD56+ non- conventional T cell subsets. Here, a combination of CD3 and TCRγδ conjugated to PerCP- Vio700 was used in one of the drop-in channels and removed from the exclusion channel covering CD14, CD19 conjugated to VioBlue and SYTOX ® Blue for dead cell exclusion (Table 3). Figure 1: Experimental design to compare variability between flow cytometers Flow chart of the experimental set up outlining the establishment and verification of the NK cell phenotype and function panels to compare variability between flow cytometers. Following selection of backbone and drop in antibodies, reference samples were used for optimizing and checking fluorochrome intensities using appropriate single stains and fluorescence minus one (FMO) controls to establish a protocol. Further, the data generated from three instruments were analysed using KALUZA ® software and results were tabulated using Graphpad Prism for statistical differences for validation using the same reference sample.