PMC:7172841 / 32320-33768 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/7172841","sourcedb":"PMC","sourceid":"7172841","source_url":"https://www.ncbi.nlm.nih.gov/pmc/7172841","text":"Peripheral blood mononuclear cells (PBMCs) were isolated after gradient centrifugation over Ficoll (Biochrom, Berlin, Germany) for 20 min at 1,400 g. After three washings in ice-cold PBS pH 7.2, PBMCs were counted in a Neubauer plate with trypan blue exclusion of dead cells. They were then diluted in RPMI 1640 enriched with 2mM of L-glutamine, 500 μg/mL of gentamicin, 100 U/mL of penicillin G, 10 mM of pyruvate, 10% fetal bovine serum (Biochrom) and suspended in wells of a 96-well plate. The final volume per well was 200 μL with a density of 2 × 106 cells/mL. PBMCs were exposed in duplicate for 24 h or 5 days at 37°C in 5% CO2 to different stimuli: 10 ng/mL of Escherichia coli O55:B5 lipopolysaccharide (LPS, Sigma, St. Louis, USA); 5 × 105 colony forming units of heat-killed Candida albicans; 25% of patient plasma; or 10 μg/mL of Tocilizumab (Roche, Athens, Greece). After incubation, cells were removed and analyzed for flow cytometry. Concentrations of TNFα, IL-1β, IL-6, IL-17A and IFNγ were measured in cell supernatants and/or serum in duplicate by an enzyme immunoassay (Invitrogen, Carlsbad, California, USA). The lowest detections limits were as follows: for TNF-α, 40 pg/mL; for IL-1β, 10 pg/mL; for IL-6, 10 pg/mL; for IL-17A, 10 pg/mL; and for IFN-γ, 12.5 pg/mL. 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