Ficoll isopaque density gradient centrifugation

For cell isolation, SF was incubated with hyaluronidase (Sigma-Aldrich, St. Louis, MO) for 30 min at 37°C to break down hyaluronic acid. SFMCs and PBMCs were isolated using Ficoll Isopaque density gradient centrifugation (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), and were used after freezing in fetal calf serum (FCS) (Invitrogen, Waltham, MA) containing 10% DMSO (Sigma-Aldrich).

Peripheral blood mononuclear cells (PBMCs) from healthy donors were separated from peripheral blood by ficoll isopaque density gradient centrifugation (GE Healthcare Bio-Sciences AB) and stained with anti-CD3, anti-CD19 and anti-CD56 antibodies. CD19+ B cells and CD3-CD56+ NK cells were isolated by FACS sorting and subsequently cultured in RPMI supplemented with 10% human serum (Sigma). For NK cell activation 1000 U/ml interleukin-2 (Proleukin) and 50 ng/ml interleukin-15 (Immunotools) were added for 18 hrs. B cells, naive NK cells or activated NK cells were harvested, washed, counted and added to neuroblastoma cells at indicated effector:target ratios for 24 hrs. For cell-cell contact experiments neuroblastoma cells were added to the lower part of the transwell system (Greiner Bio-one, 1 um pore size) and immune cells were added to the upper part and cultured for 24 hrs. For blocking experiments the NK cells were mixed with anti-IFNγ (BD Biosciences) or anti-IP10 (R&D) neutralizing antibodies (1 ug/ml) and subsequently added to the neuroblastoma cells.

Peripheral blood mononuclear cells (PBMCs) were isolated from the blood of healthy donors by ficoll isopaque density gradient centrifugation (GE Healthcare Bio-Sciences AB) and frozen until use. PBMCs were used as a source of monocyte-derived dendritic cells (MoDCs). Immature DCs were cultured as previously described [23 (link)]. Briefly, PBMCs were seeded in standard 48- or 96-well culture plates in X-VIVO-15 medium with gentamycin (Lonza), supplemented with 2% heat-inactivated and 0.2 μm-filtered FCS (Bodinco). Cells were allowed to adhere for 1 h. Non-adherent cells were subsequently removed by washing with PBS and adherent cells were cultured in serum-free X-VIVO-15, supplemented with 450 U/mL GM-CSF and 300 U/mL IL-4 (Milteny). Medium was refreshed on the second day of incubation. On day 5, cells were stimulated with LPS, NSR or NSRmock diluted in RPMI 1640 with HEPES and glutamine (Gibco), supplemented with 10% FCS. Medium was used for negative controls. The stimulation conditions were selected for optimal infectivity of NSR. DCs were harvested at different time points after stimulation as indicated in the results section, by replacing the growth medium with cold PBS, followed by shaking (450 rpm) of the culture plates for 1 h at 4°C to detach cells.