Ficoll paque plus density

Peripheral blood mononuclear cells (PBMCs) were collected from healthy voluntary donors and isolated from the whole blood via Ficoll-Paque PLUS density gradient centrifugation (cat# 17144002, cytiva). CD3⁺ T cells were isolated using the Pan T cell isolation kit (cat# 130-096-535, Miltenyi Biotec). Next, the isolated CD3⁺ T cells were stimulated for 48 h with the human T cell activation/expansion kit (cat# 130-091-441, Miltenyi Biotec) in accordance with the manufacturer’s instructions. For this process, the T cells were cultured in X-VIVO™ 15 media (cat# 02-060Q, Lonza) supplemented with 10% inactivated FBS, 10 ng/mL human IL-7 (cat# 200-07, Peprotech), and 10 ng/mL human IL-15 (cat# 200-15, Peprotech) at a final concentration of 1 × 10⁶ cells/mL.

Peripheral blood mononuclear cells (PBMCs) from 12 voluntary and healthy HCMV- and EBV-seropositive blood donors, were isolated from buffy-coats by Ficoll-Paque PLUS density (Cytiva) gradient centrifugation according to the manufacture’s instruction. CD56⁺ NK cells were then enriched by magnetic labelling using the human CD56⁺ NK cell Isolation Kit according to the manufacturer’s instruction (Miltenyi Biotec). NKG2C^-NKG2A⁺, NKG2C⁺NKG2A^- and NKG2C⁺NKG2A⁺ NK cells were then sorted on a FACSAria Fusion (BD Bioscience). Sorted cells were stored frozen at −80 °C in 1x10⁶ viable cell per aliquots in 90% FCS + 10% DMSO (both: Thermo-Fisher).