The anti-human monoclonal antibodies used for immunostaining were Alexa-Fluor®647 anti-CD120a/TNFR1 (clone H398; AbD Serotec), Alexa-Fluor®700 anti-CD11c, e-Fluor®450 anti-CD123 (clones 3.9 and 6h6 respectively; eBioscience), FITC and PE anti-HLA-DR (clone L243, BioLegend), PerCP-Cy5.5 anti-CD14 and Pacific Blue™ anti-CD16 (clones HCD14 and 3G8 respectively; BioLegend) and FITC Lin-1 cocktail (BD Biosciences). Alexa-Fluor®647 mouse IgG2a (AbD Serotec) was used as an isotype control for CD120a/TNFR1 staining. Data obtained were analyzed using FlowJo (Tree Star, Inc.). TNFR1 surface expression was calculated by dividing the median fluorescence intensity (MedFI) of the anti-TNFR1 staining by the MedFI of the isotype control for each sample. Each day that donor samples were evaluated, FluoroSpheres were also analyzed (Blank Beads and Calibration Beads, Dakocytomation). Data normalization was performed using the Calibration Beads to control for day-to-day variation in flow cytometer function.
