Anti human cd14 clone m5e2

Intracellular expression of TLR8 was assessed on T cells, B cells, and monocytes by flow cytometry. Cells collected into sodium heparin tubes and peripheral blood mononuclear cells (PBMCS) were isolated by Ficol gradient and incubated with fluorochrome-conjugated anti-human CD3 (clone SK7), anti-human CD19 (clone HIB19), and anti-human CD14 (clone M5E2, all from BD Biosciences, San Diego, CA, USA) to identify T cells, B cells, and monocyte populations, respectively. To determine intracellular expression of TLR8, cells were fixed and permeabilized (BD Cytofix/Cytoperm, BD Biosciences) following manufacturer instructions and blocking with 2% pooled human serum for 20 min at 4 °C in the dark and intracellularly stained with the primary antibody (mouse anti-human anti-TLR8 (clone 44C143, Acris Antibodies, Herford, Germany) or mouse IgG2a isotype control antibody (eBioscience, San Diego, CA, USA) for 30 min followed by polyclonal goat-anti-mouse fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Dako) for another 30 min. The cells were washed and acquired in FACS-Canto (BD Biosciences) cytometer. The expression of TLR8 was gated and analyzed (FACSDiva Software; BD Biosciences) as mean fluorescence intensity (MFI) with regard to the isotype control signal. See Figure S1A for the gating strategy and Figure S1B for backgating.

MDDC were matured and then stained for markers of maturation and activation (anti-human CD14 clone M5E2, CD86 clone 2331 (FUN-1), CD80 clone L307.4, HLA-DR clone TU36, CD83 clone HB15e, CD40 clone 5C3, CD197 clone 3D12, and CD11c clone 3.9) (BD Bioscience).