DC maturation and flow cytometry.

The dendritic cell (DC) maturation experiments were contracted to and run by AllCells (Alameda, CA) using standard company protocols. Briefly, human PBMCs from 3 separate donors were cultured in RPMI medium supplemented with 10% FBS, 100 ng/ml human IL-4, and 140 ng/ml human granulocyte-macrophage colony-stimulating factor (GM-CSF) for 7 days, with medium supplementation on day 3. After the 7-day differentiation period, DCs were stimulated with BECC-derived TLR4Ls for 24 h at 37°C, 5% CO₂. Cells were then lifted off the culture well using Accutase reagent, and EDTA added to halt enzymatic digestion. Cells were washed twice with PBS, nonspecific binding was inhibited using FcBlock (Miltenyi), and finally, cells were stained with CD80-fluorescein isothiocyanate (FITC), CD40-phycoerythrin (PE), and CD83-PE (BD Biosciences). Flow cytometry analysis was performed using an LSRII (BD Biosciences). Dead cells/debris were excluded using a forward scatter/side scatter (FSC/SSC) gate, 10,000 events were collected for each sample, and isotype controls for each antibody were run and determined to contain <1% of events recorded.