Ficoll hypaque gradient

PBMCs were collected from 2 times diluted blood sample after centrifugation at 2000g for 30 min upon 5 ml Ficoll-Hypaque gradients (Sigma, USA). The PBMCs obtained were washed twice with ice-cold PBS and suspended in RPMI 1640 containing 10% fetal calf serum (FCS). Then, PBMCs were cultured in a 5% CO₂ incubator at 37°C.

Peripheral blood mononuclear cells (PBMCs) were separated from fresh blood samples by Ficoll-Hypaque gradients (Sigma-Aldrich, St. Louis, MO, USA) and processed within 4 h. The reconstitution of NK cells after HSCT was determined by eight-color multiparameter flow cytometry using the following mAbs: anti-CD45-FITC (clone; HI30, BD bioscience, San Diego, CA, USA), anti-CD3-V450 (clone; UCHT1, BD bioscience, San Diego, CA, USA), anti-CD16-V500 (clone 3G8; BD bioscience, San Diego, CA, USA), anti-CD56-PE-Cy7 (clone; B159, BD bioscience, San Diego, CA, USA), anti-NKGD-APC (clone; 1D11, BD bioscience, San Diego, CA, USA), anti-NKG2A-PE (clone; REA110, Miltenyi Biotec, Bergisch Gladbach, Germany), anti-NKG2C-Alexa700 (clone 134591, R&D Systems Inc., Minneapolis, MN, USA), and anti-CD57-V450 (clone; TB01, ebioscience, San Diego, CA, USA). PBMCs were also intracellularly stained with anti-CD3ζ-PE (clone; 6B10.2, BD bioscience, San Diego, CA, USA) and anti-FcεRIγ-FITC (FcRγ) (Millipore, Merck Millipore, Burlington, MA, USA) for the analysis of FcεRIγ-deficient NK cells (g-NK cells). NK cell gating and analyses of the NK subpopulation are shown in Figure A1. Flow cytometry data were collected on an FACS Fortessa instrument (BD bioscience, San Jose, CA, USA), using FlowJo version 10.0.6 software (Tree Star, Ashland, OR, USA).

Peripheral blood mononuclear cells (PBMCs) were isolated from EDTA-anticoagulated peripheral blood by centrifugation on Ficoll-Hypaque gradients (Sigma-Aldrich). For surface staining, antibodies specific for CD4 (labelled with FITC or Pacific Blue), CD28 (PE or APC), NKG2D (PE), CD11a (PE), CD62L (PECY7), CX3CR1 (APC) and CD49d (PE) were purchased from Biolegend (San Diego, USA). For characterization of adhesion molecules, integrins and chemokine molecules, PBMCs from the healthy controls and LN patients were seeded in 96-well round-bottom plates at 1 × 10⁶ cells/well in culture medium and stimulated with 50ng/ml recombinant human IL-15 (R&D Systems, Minneapolis, USA). After 48-72 h culture, cells were stained with the corresponding antibodies. PBMCs were stimulated with anti-CD3 (1µg/ml), IL-15 (50ng/ml) or both, and cultured for 24 h for NK receptor analysis and 72 h for cytotoxic molecule analysis. Cells were surface stained or intracellularly stained with anti-NKG2D-PE, anti-perforin-PE, anti-granzyme B-APC and anti-IFN-gamma-PE. All samples were analyzed by flow cytometry using an LSR II (BD Biosciences) or a Cyan flow cytometer (Beckman, CA, USA).

The MYC-N amplified neuroblastoma (NB) cell line HTLA-230 was provided by Dr. E. Bogenmann (Children’s Hospital Los Angeles, CA) (Corrias et al., 1996 (link)) and cultured in RPMI-1640 medium supplemented with 10% heat-inactivated FCS (Biochrom, Berlin, Germany), 50 mg/ml streptomycin, 50 mg/ml penicillin (Sigma-Aldrich), and 2 mm glutamine (Euroclone). The cells were cultured in a humidified environment (95% air/5% CO₂) at 37°C and were used to generate 3D tumor models.
Peripheral blood mononuclear cells (PBMCs) were obtained from blood of volunteer healthy donors by Ficoll-Hypaque gradients (Sigma Aldrich). NK cells were purified by using the NK-cell isolation kit II (Miltenyi Biotec) and were cultured on irradiated PBMCs in RPMI-1640 supplemented with 10% heat-inactivated FCS, 50 mg/ml streptomycin, 50 mg/ml penicillin (Sigma-Aldrich), 2 mm glutamine (Euroclone), 600 IU/ml rhIL-2 (Proleukin; Chiron, Emeryville, CA) and 0.5% v/v phytohemagglutinin (Gibco, Paisley, United Kingdom). After 10 passages, NK cells were checked for purity (>95%) analyzing classical NK cell markers (Castriconi et al., 2007b (link)).