Cd16 alexa 700

Patient samples were collected in an EDTA tube between 8 am and 2 pm, and were analyzed within 6 hours of collection. RBCs were removed from samples using Pharmlyse (BD Biosciences), and white blood cells were stained for flow cytometric analysis. Stains included a permeability exclusion dye (Thermofisher) as well as CD11b PE-Cy7 (clone ICRF44, Biolegend), CD64 PerCP-Cy5.5 (clone 10.1, BD Biosciences), CD16 Alexa 700 (Clone B73.1, Biolegend), CD14 V450 (Clone MoP9, BD Bioscience), and CD45 APC-Cy7 (Clone 2D1, Biolegend); gating is described in the results section and shown in Fig. S2, available as supporting information in the online version of this paper. Samples were acquired on a BD LSR-II using FACSDiva Software, and the data were analyzed using FlowJo Version X software.

Poly I:C, Gardiquimod, ssRNA40, PolyI:C/Lyovec and Lyovec (vehicle control) were purchased from Invivogen (San Diego, CA). Human antibodies; CD16 APC, CD16 FITC, CD14 FITC, CD40 FITC and CD86 FITC were purchased from eBioscience (San Diego, CA). Antibodies CD14 APC, CD14 PE, CD40 PE-Cy7, CD163 PE, CD11c APC, CD68 PE, CD206 APC, DC-SIGN-FITC and isotype control antibodies were purchased from BD Pharmingen (Franklin Lakes, NJ). Human antibodies CD40 Alexa 700, CD68 PE-Cy7, Brilliant violet LAP (TGFβ), CD206-PE, CD16 Alexa-700 were purchased from Biolegend (San Diego, CA). Transwell-6 system with a 0.4-μm porous membrane was purchased from BD Biosciences, Franklin Lakes, NJ. TLR3, TLR7, TLR8 siRNA and scrambled siRNA were purchased from Ambion Life Technologies (Carlsbad, CA).