Anti cd14 clone hcd14

PBMCs were thawed, stained with LIVE/DEAD Green Viability Dye (Invitrogen) for 15 minutes at 4°C and subsequently preincubated for 10 min with of FcR blocking reagent (Miltenyi). Afterward, cells were incubated for 25 minutes at 4°C with anti-CD3 (clone HIT3a; BioLegend), anti-CD14 (clone HCD14; BioLegend), anti-CD19 (clone HIB19; BioLegend), anti-CD56 (clone HCD56; BioLegend) and anti-CD16 (clone 3G8; BioLegend) antibodies. Cells were then sorted for CD56^dim CD16⁺ cNK cells and CD56^bright CD16^- NK cells on a BD FACSAria Fusion (BD Bioscience) at the Ragon Institute Imaging Core Facility.

Flow cytometry was used to analyze surface expression of adhesion molecules on human neutrophils. Cells were isolated using a Histopaque^® density gradient (Sigma Aldrich, Taufkirchen, Germany), washed and incubated with according anti-human antibodies. Antibodies included: anti CD11b, clone M1/70; anti CD14, clone HCD14; anti CD15, clone HI98; anti CD49d, clone 9F10 (all from BioLegend, San Diego, CA, USA); anti CD182, clone 6C6; anti CD 29, clone HUTS-21; anti CD162, clone KPL-1 (all from BD Bioscience, Franklin Lakes, NJ, USA); anti CD11a, clone MEM-25; anti CD62L, clone LT-TD180 (all from ImmunoTools, Friesoythe, Germany). Samples were measured on a BD FACS Canto 2 flow cytometer and data were analyzed using FlowJo v7.1.