Cd16 apc cy7 3g8

PBMCs were rested after thawing for 4 h in RPMI 1640 medium (Thermo Fisher Scientific) without supplements and subsequently incubated in RPMI 1640 containing live/dead near-infrared dye (Life Technologies) for 20 min at room temperature. Cells were washed, split into two wells, and resuspended in either only RPMI 1640 for the unstimulated sample or RPMI 1640 containing 10 µg/ml goat anti-human IgG/IgA/IgM f(ab)2 (Jackson ImmunoResearch). Stimulation was done for 5 min including a 2-min centrifugation step to remove the medium, followed by addition of the fixation and permeabilization solution of the Foxp3 staining kit according to the manufacturer’s instructions (eBiosciences). Cells were fixed and permeabilized for 45 min at 4°C and then stained with antibodies including CD3 APC-Cy7 (SK7; BioLegend), CD10 PE-Cy5 (HI10a; BD Biosciences), CD14 APC-Cy7 (HCD14; BioLegend), CD16 APC-Cy7 (3G8; BioLegend), CD19 BV510 (SJ25C1; BD Biosciences), CD21 BV711 (B-ly4; BD Biosciences), CD27 PE-CF594 (M-T271; BD Biosciences), IgD APC (IA6-2; BioLegend), and total phosphor-tyrosine PE (PY20; BD Biosciences) for 30 min at room temperature. Cells were extensively washed and measured using a BD Fortessa. Analysis was done with FlowJo 10 (TreeStar).