Ab serum

Heparinized blood samples from 19 AAD patients (see Table 1 for patient details) and 21 controls were processed essentially as described previously (Bratland and others 2013 (link)). In brief, plasma samples were isolated, aliquoted, and kept frozen at −20°C, while PBMC were isolated using Ficoll-Paque Plus (GE Healthcare). Upon isolation PBMC were kept cryopreserved at −150°C in 90% AB serum (Lonza) and 10% dimethylsulphoxide (DMSO). PBMC stored at −150°C were thawed, washed, and resuspended in serum-free AIM V medium (Life-technologies). The cells were seeded at 1×10⁶ cells in 500 μL medium in 24-well culture plates, and stimulated with various IFNs or polyinosinic:polycytidylic acid (poly (I:C)) depending on the total number of cells available: IFN-α (PBL Biomedical laboratories) at 10⁴ U/mL, IFN-β (RnD Systems) at 10⁴ U/mL, IFN-γ (Biolegend) at 1 μg/mL, and poly (I:C), (Invivogen) at 10 μg/mL. Optimal doses for each stimulus were determined in preliminary experiments. Nonstimulated cells grown in medium alone served as controls. The concentrations of chemokines or IFNs induced by medium alone were subtracted from the IFN- or poly (I:C)-induced levels. The cells were grown for 24 h at 37°C with 5% CO₂ in a humidified incubator, upon which cells and supernatants were harvested and stored at −80°C for RNA isolation and downstream assays, respectively.

PBMCs were isolated by density gradient separation using LymphoPrep (cat #1114547; Axis-Shield, Oslo, Norway) at 1200 x g for 30 minutes, washed twice in phosphate buffered saline followed by centrifugation at 400 x g, and re-suspended in RPMI 1640 (cat #01-106-1a; Biological Industries, Kibbutz Beit Haemek, Israel) containing 30% (v/v) AB serum, 25 mM HEPES, 2 mM L-glutamine (cat #25030–024; Gibco, Life Technologies, Carlsbad, CA), and 50 μg/mL gentamycin sulfate (cat #03-035-1c; Biological Industries). All subsequent centrifugations were carried out at 400 x g. Human serum isolated from healthy male donors of blood group AB (AB serum) was purchased from Lonza (cat # 14-490E; Basel, Switzerland) and used as the serum source in all experiments.

Twenty mL of peripheral blood were obtained by venipuncture from the 20 selected patients of Group 2 patients and collected into sterile EDTA tubes. The PBMCs were immediately separated by density gradient centrifugation over Ficoll–Hypaque (Lonza, BioWhittaker) and then washed twice with RPMI 1640. Cell count and viability were determined utilizing Guava ViaCount Flex Reagent for Flow Cytometry (Merck Millipore, France). Viability was exceeding 95% in all studied cases. PBMCs were suspended in RPMI 1640 medium, supplemented with 2 mM l-glutamine, 25 mM HEPES, 100 U/mL benzyl-penicillin, 0.1 mg/mL streptomycin, and 10% ABserum (complete medium) (Lonza, BioWhittaker). All cultures were incubated.