Dylight 633

FVIII (50 nM, Helixate®), FVIII^HSQ (50 nM), FVIII^C1 (50 nM), ADAMTS-13 (50 nM) conjugated to Dylight 633 (Thermo Scientific, Courtaboeuf, France) or human Factor IX (50 nM, Benefix, Pfizer, France) were incubated with 20% normal serum (NS), heat-inactivated (56°C for 30 min) serum (HIS) or C3-depleted serum (ΔC3) in RPMI-1640 at 37°C for 1 hour (h). When indicated, FVIII or Dylight 633-labeled ADAMTS-13 were incubated with C3 (250 μg/mL), Factor B (50 μg/mL) and Factor D (1 μg/mL) or with C3b (250 μg/mL) in 20 mM HEPES 150 mM NaCl 10 mM MgCl2 for 1 h at 37°C. Samples were then incubated with 5-day-old immature MO-DCs (2.10⁵ cells/100 μL) for 2 h at 4°C or 37°C. Cells were washed with ice-cold phosphate buffer saline, fixed with 4% paraformaldehyde (Sigma-Aldrich). In the case of FVIII, cells were permeabilized using 0.5% saponin (Sigma-Aldrich) and stained with an FITC-conjugated monoclonal mouse anti-human FVIII IgG (77IP52H7). Cells were analyzed by flow cytometry and data were computed using the BD FACS Diva software (v.6.1, BD Biosciences). The uptake was quantified as the difference in the median fluorescence intensities between 37°C and 4°C (ΔMFI_37°C–4°C) to exclude the signal generated by the binding of FVIII to the cell surface.