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40 | Chapter 2 Separately gated NK cells (CD56+/CD3-), T cells (CD3+CD56-) and CD3+CD56+ cells (within the CD45 + gate) were then analysed for degranulation 24 (by CD107a), ADCC potential (by CD16) 25 , and activation status (by CD25, NKp44) 26,27 (Table 3 and Supplementary Table S3 online). By developing this standardized and harmonized gating strategy a solid basis was set to check the individually developed acquisition protocols of all three participating centers for inter-center variability. Table 2: Antibody specifications - NK phenotype panel Laser Antibody Fluorochrome Clone Titration Manufacturer Catalogue No Violet 405nm CD45 VioGreen  5B1 1:11 Miltenyi 130-096-906 CD3 VioBlue  BW264/56 1:11 Miltenyi 130-094-363 TCRγδ VioBlue   11F2 1:11 Miltenyi 130-101-557 CD14 VioBlue  TÜK4 1:11 Miltenyi 130-094-364 CD19 VioBlue  LT19 1:11 Miltenyi 130-098-598 Sytox® Blue Dead cell marker  1:1000 Life technologies S11348 Table 1: Comparison of instrument settings between three flow cytometers from three centers participated in the study Fluoro chromes Filter settings - Band pass & Long pass filters PMT voltages BD FACS Canto™ II BD LSR Fortessa™ MACS Quant® Analyzer X BD FACS Canto™ II BD LSR Fortessa™ MACS Quant® Analyzer X FSC 488/10 488/10 488/10 319 489 328 SSC 488 /10 488/10 488/10  462 266 476 FL1 530/30 & 502LP 530/30 & 505LP 525/50 496 484 417 FL2 585/42 & 556LP 575/26 & 550LP 585/40  435 473 416 FL3 780/60& 735LP 780/60& 750LP 750LP  540 549 487 FL4 670LP& 655LP 695/40& 685LP 655-730 507 681 594 FL5 660/20 670/14& 655LP 655-730 588 484 522 FL6 780/60& 735LP 780/60& 750LP 750LP 474 451 579 FL7 450/50 450/50 450/50 375 423 444 FL8 510/50& 502LP 525/50& 505LP 525/50 403 453 560 Three flow cytometers (BD FACS Canto™ II, BD LSR Fortessa™ and MACS Quant® Analyzer X) with compatible optical configuration were used for NK phenotype and NK function FACS panel design and optimization studies. Forward scatter (FSC), side scatter (SSC) and eight fluorescence emission channels (FL1-FL8) were used. Further, their corresponding band pass and long pass (LP) filter settings were compared and the photomultiplier tube (PMT) voltages obtained were mentioned in table 1.