Facs canto 2 with facsdiva software

Fresh harvested PBMCs and monocytes were seeded in a polypropylene 96-well plate (0.2×10⁶ cells per well) and washed twice with flow cytometry staining buffer (PBS containing 0.5% endotoxin-free BSA, 2mM EDTA, and 0.1% NaN₃ (Merck Millipore, 1687)). Cells were incubated with a mix of cell-specific antibodies (phycoerythrin anti-CD3 antibody (Ebioscience), fluorescein isothiocyanate-conjugated anti-CD66b, allophycocyanin anti-CD14, phycoerythrin cyanin-7 anti-CD56, and Alexa Fluor 700 anti-CD16 antibodies (BD Biosciences)), for 30 min at 4°C in the dark. Before analysis by FACS, cells were washed three times as described above and resuspended in a total volume of 300 μl FACS buffer. Monocyte purity was verified via flow cytometry (FACS Canto II with FACSDiva Software; BD Biosciences, Heidelberg, Germany).

Heparin anticoagulated blood was collected within 24 h of hospital admission, and one month thereafter. Polymorphonuclear cells were isolated from the lowest fraction of Ficoll-Paque separated heparin tubes, and erythrocytes were lysed with erythrocyte lysis buffer (Sigma-Aldrich, Saint Louis, Missouri). Cell purity was 94.1% [IQR, 86.0–97.3] as determined by flow cytometry (FACS Canto II with FACSDiva Software; BD Biosciences, Heidelberg, Germany) using anti-CD14-APC, anti-CD3-PECy7, and anti-CD66-FITC (BD Bioscience, San Jose, CA). See Figure S1 for the gating strategy.