Cd45 hi30

Single cell suspensions were stained with fixable viability dye eFluor^® 780 (eBioscience) for 30 min at 4°C, and Fc receptors were blocked with anti-CD16/32 (BioLegend) blocking antibody prior to surface staining with antibodies. Antibodies for surface staining used are listed, mouse: CD45 (30-F11, BioLegend), TCR β (H57-597, BioLegend), CD69 (H1.2F3, eBioscience), CD1d tetramer (NIH); human: CD45 (HI30, eBioscience), TCRα/β (IP26, BioLegend), CD69 (FN50, BioLegend), Vα24 (6B11, BD Bioscience). For intracellular staining, single cell suspensions were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma-Aldrich), 1 μg/mL ionomycin (Sigma-Aldrich) and 1 μg/mL Golgi stop A (BD Biosciences) for 4 h. After surface staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (BD biosciences) and further stained intracellularly with human IFNγ (B27, BioLegend) and human IL-4 (MP4-25D2, BioLegend). For Ki67 staining, cells were fixed and permeabilized with Cytofix/Cytoperm solution (eBioscience), and further stained with Ki67 (SolA15, eBioscience). Data were acquired using LSR II (BD Biosciences) and analyzed using the FlowJo software (BD Biosciences).

Whole blood was fixed with 16% formaldehyde (Thermo Scientific) for 10 min at room temperature. Pre-warmed lysis and permeabilization buffer (0.114% Triton X-100 in PBS) was then added and the samples incubated for 15 min at 37 °C. The cells were then washed in cold wash buffer (4% FBS in PBS), and re-suspended in ice-cold 50% MetOH in PBS for 10 min at 4 °C. The cells were then washed with cold wash buffer, and then stained with anti-CD14 [RMO52], (#A22331, IOTest), anti-CD16 [B73.1] (#12-0167-42, eBioscience), CD45 [HI30] (#45-0459-42, eBioscience) and anti-p-ERK1/2 (pT202/pY204) [20A] (#562644; BD Biosciences) or anti-p-p65 (p-S529) [K10-895.12.50] (#558422; BD Biosciences) in wash buffer for 30 min at room temperature.

For phenotypic analysis, cells were labelled with Abs recognizing CD11c (3.9), CD40 (5C3), CD45 (HI30), CD83 (HB15e), HLA-DR (LN3, all eBioscience), BDCA1/CD1c (AD5-8E7, Miltenyi Biotec), BDCA3/CD141 (AD5-14H12, Miltenyi Biotec), CD86 (2331, BD Horizon), CLEC9A (8F9, BioLegend), HBsAg (Acris), a lineage cocktail including CD3 (UCHT1, eBioscience), CD14 (61D3, eBioscience), CD19 (HIB19, eBioscience) and CD56 (MY31, BD Biosciences), and the live/dead marker Aqua (LifeTechnologies). Fluorescence was measured using a FACS Canto II (BD Biosciences).