NK Cell Degranulation Assay with PBMC

On day 6 PBMC, either GAD65 AA 114–122 or FLU peptides stimulated for 4 days, additionally cultured for two days in the presence of IL-2 (100 IU/ml), were co-cultured with pulsed or unpulsed APCs at 1:3 (PBMC:APCs) ratio in 96 well round bottomed culture plates (Corning Incorporated, Corning, NY 14831–001, USA) for 2 and half hours in RPMI 10% FBS complete medium (see above) additionally supplemented with GolgiStop reagent (1:500 dilution, BD Biosciences). An experimental positive control was set-up by NK cell isolation from PBMC of a HD volunteer previously obtained with the RosetteSep method (Stem Cell Technologies, Vancouver, Canada) and FicollPaque Plus (Lympholyte, Cedarlane Laboratories, Burlington, Ontario, USA). Isolated NK cells have been routinely checked for the percentage of CD3^-CD56⁺ cells by FACS analysis and those with purity greater than 90% were cultured with 200 IU/ml of recombinant human IL-2 (Sigma-Aldrich) at 37°C and used up to 5 days after isolation as effectors in degranulation assay. Isolated NK cells were then co-cultured with K562 cells (American Type Culture Collection, ATCC), a tumoral cell line known to induce NK cell degranulation according to standard protocols, as control target [41 (link)], and with either GAD65 AA 114–122 peptide pulsed and unpulsed APCs.