Anti human lineage cocktail

PBMC from healthy human donors (Interregionale Blutspende SRK) were isolated using Ficoll (GE Healthcare, Chicago, IL, USA) density gradient centrifugation. Monocytes were enriched using the EasySep^TM Human Monocyte Enrichment Kit w/o CD16 Depletion (STEMCELL Technologies).
DC were isolated using the MACS-based Pan-DC Enrichment Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) followed by Fc-receptor blocking with anti-mouse CD16/32 (2.4G2, generated in house) for 15 min. Subsequently, cell surface markers were stained using anti-CD11c (3.9), anti-CD123 (6H6), anti-human lineage cocktail (CD3/14/16/19/20/56), anti-CD141 (M80), anti-HLA-DR (L243) (Biolegend, San Diego, CA, USA) in FACS buffer (PBS with 2% FBS and 1 mM EDTA) for 45 min on ice. Cells were then purified by FACS using the Moflo Astrios EQ cell sorter (Beckman Coulter, Nyon, Switzerland). Ethical approval provided by the Kantonale Ethikkommission Bern (KEK), Switzerland. Code: 2017-02246.

The following fluorescently conjugated antibodies were used for phenotypic analysis of NK cells: anti-LFA-1 (363404); anti-CXCR3 (353720); anti-PD-1 (329906); anti-CD107a (328612); anti-NKP44 (325116); anti-CD158e1 (312706); anti-CD158d (347006); anti-CD27 (356412); anti-CCR4 (359412); anti-DNAM-1 (338316); anti-CD16 (302040); anti-Granzyme B (515406); anti-CD62L (304806); anti-CD69 (310910); anti-NKP80 (346706); anti-NKP30 (325210); anti-CD158f (341304); anti-CD158b (312612); anti-Tim-3 (345012); anti-CD94 (305504); anti-TIGIT (372706); anti-TRAIL (308206); anti-CD57 (322306); anti-CX3CR1 (341610); anti-NKG2D (320808); anti-Perforin (353310); anti-Ki67 (350504); anti-IFN-γ (506518); anti-CD94 (305506); anti-NKp46 (331916); anti-CTLA4 (369614); anti-CD96 (338416); anti-41BB (309818); anti-CD25 (356108) were purchased from Biolegend. anti-CD159a/NKG2A (FAB1059P) and anti-NKG2C (FAB138G) were purchased from R&D.
The following fluorescently conjugated antibodies were used to identify immune cell types in eNK or PBMC: anti-human Lineage Cocktail (348803); anti-CD56 (362550); anti-CD3 (300430); anti-CD33 (366620); anti-HLA-DR (307606); anti-CD14 (301836); anti-CD19 (302242); anti-CD11b (301322); anti-CD25 (356108); and anti-FOXP3 (320108) were purchased from Biolegend, San Diego, CA, USA.

Antibodies conjugated with FITC, PE, PerCP, APC, AF700 and Pacific blue against the surface on intracellular molecules CD3, CD4, CD8, CD16, CD25, CD56, CD69, CD95, CD123, CD178, CD197, CD45RA, HLA-DR, CD11c, CD11b, CD127, Foxp3, Perforin, Granzyme B, IFN-γ, IL-17 as well as the anti-human lineage cocktail, CD3,CD14,CD19,CD20, CD56 including isotype-matched controls were purchased from Biolegend (Nordic Biosite, Täby, Sweden) and eBioscience, (San Diego, CA, USA). PMA and ionomycin were purchased from Sigma (St Louis, MO, USA) and Brefeldin A was obtained from BD (Mountain view, CA, USA).