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54 | Chapter 2 volumetrically by FACS using a cocktail of 7-AAD (1:200) (Sigma Aldrich, Zwijndrecht, The Netherlands), CD3 VioBlue (1:11), CD56 APC-Vio 770 (1:11), CD16 APC (1:11), CD45 VioGreen (1:11), and CD25 VioBrightFITC (1:11) antibodies from Miltenyi Biotec GmbH. PBMC viability (7AAD- CD45+) and NK cell (7AAD- CD45+ CD3- CD56+) and NK CD16 (7AAD- CD3- CD56+ CD16+) percentages measured before and after overnight incubation were 7AAD- CD45+ (lymphocytes): 91% ± 4% versus 84 ± 2%, %; 7AAD- CD45+ CD3- CD56+ (NK cells): 7.1% ± 3% versus 6.8% ± 6% and CD3- CD56+ CD16+ (CD16 positive cells within NK cells): 89% ± 3% & 82% ± 9%. Cryopreservation and thawing of PBMC PBMC were suspended at 1x 10 7 viable cells/ml in 500µl human serum albumin (HSA) per tube and 500µl of freezing medium containing 14% Dimethyl Sulfoxide (DMSO), (Sigma Aldrich, Zwijndrecht, The Netherlands) in Roswell Memorial Park Institute RPMI medium (Sigma Aldrich, Zwijndrecht, The Netherlands) was added to the cells. Isolated PBMC were frozen at a final concentration of 7% DMSO in pre-cooled Nalgene ® Mr. Frosty freezing containers (Thermo Scientific, Landsmeer, The Netherlands) overnight at -80°C and transferred after 24h to liquid nitrogen for longer storage. To thaw PBMC, cryovials were transferred from liquid nitrogen into a pre-warmed 37°C water bath. Cells were thawed and washed twice in 500µl of PBS + 0.5% HSA to remove toxic DMSO and suspended in RPMI medium containing 2% HSA for further studies. Sample handling In the first phase of the study, peripheral blood mononuclear cells (PBMC) from one healthy donor were collected at different time points as reference samples (n=6) to establish harmonized data acquisition and analysis between three participating centers. All reference samples were isolated and cryopreserved at one center and shipped to other participating centers (n=3). The cryopreserved samples were thawed and analysed at least one month after PBMC collection at all participating centers. At all centers same 3-time points of reference samples were used to optimize the instrument settings, staining protocols and panels. An additional 3-time points of the same reference sample were stained with the NK cell phenotype panel antibody mix to show the obtention of reproducible data sets across the 3 centers. In the second phase of the study in which the NK cell phenotype and function was compared between fresh and cryopreserved NK samples, PBMC were collected from different healthy donors (n=12; 6 samples were processed at Miltenyi Biotech and analysed using a MACSQuant ® Analyzer X device, and other 6 were processed at VU medisch centrum and analysed using BD LSRFortessa TM . One fraction of the isolated PBMC was cryopreserved and another fraction was used directly for NK cell analysis. Further, for testing differences