Verikine human ifn α multi subtype serum elisa kit

Supernatant cytokine concentrations were determined with Bio-Plex Pro magnetic multiplex assays (Bio-Rad; Hercules, CA) and analyzed on the Bio-Plex 200 system with Bio-Plex Manager 5.0 software (Bio-Rad), which uses the Brendan five-parameter logistic regression for standard curve fitting. Interferon (IFN)-α concentrations were measured by enzyme-linked immunosorbent assay with the VeriKine Human IFN-α Multi-Subtype Serum ELISA kit (PBL Assay Science; Piscataway, NJ). Cytokine concentrations were expressed as a percentage compared to TLRAs alone, which were defined as 100%.

Human whole blood from healthy donors was treated with either HCQ (Abcam Cambridge, MA, USA) or 24F4A (Biogen, Cambridge, MA, USA) or in combination and incubated for 30 min before treatment with TLR agonists CpG-A (10 μM), R848 (1 μM) or ssRNA (9.2 s ssRNA AGCUUAACCUGUCCUUCAA and A20 control ssRNA AAAAAAAAAAAAAAAAAAAA (4 μg/ml) for 18 h at 37 C and 5% C0₂ (Sigma-Aldrich St. Louis, MO, USA). Single stranded RNA (200 ng) was complexed with pL-Arginine (360 ng) for 15 min at room temperature in PBS (27 (link)). For the purpose of IFNα secretion from human pDCs, 9.2 ss RNA was used as a TLR7 agonist (28 (link)). Serum IFNα was measured using VeriKine Human IFNα Multi-Subtype Serum ELISA Kit from Pbl Assay Science (Piscataway, NJ, USA).

Human Peripheral Blood Mononuclear cells (hPBMCs) were collected from healthy adult donors. hPBMCs were cultured in round-bottom 96-well plates (BD Falcon, Durham, NC, USA) with a density of 5 × 10⁵ cells/well. Plated cells were pretreated with DMSO or 2.5 μM inhibitors for 3 h and then stimulated by indicated ligands, such as R848 or TLR1/2 specific ligand Pam₃CSK₄ (Invivogen, HongKong, China), for 20 h in 200 ml of RPMI 1640 medium (Gibco, Paisley, UK) supplemented with 10% FBS, Penicillin-Streptomycin-Glutamine (Gibco, Paisley, UK), 50 μM 2-ME medium at 37 °C in the presence of 5% CO₂. Supernatants were collected to measure secreted cytokines by ELISA. Concentration of human IFN-α and TNF-α in the supernatants was measured with Verikine Human IFN-α Multi-Subtype Serum ELISA Kit (PBL Assay Science, Piscataway, NJ, USA) and Human TNF-α Uncoated ELISA (ThermoFisher Scientific, Carlsbad, CA, USA), respectively. This study was approved by the Research Ethics Committee of IMSUT (accession number: 30-100-B20190419).