Human ifn α

Human PBMCs or mouse splenocytes were cultured at 0.2–1×10⁶ cells/well in 96-well U-bottomed plates using different concentrations of CpG ODNs. After 16 h, the supernatants were harvested and assayed with the following ELISA kits: human IFN-α, mouse IL-6, mouse TNF-α (Mabtech, Sweden), and mouse IFN-α (eBioscience, Vienna, Australia). Human IL-6 and TNF-α were analyzed using CBA (BD Biosciences, San Jose, CA, USA). All kits were used according to the manufacturers’ instructions and the results obtained were expressed as picogram per milliliter (pg/ml). For the CBA assays, 50 μl of diluted samples or recombinant standards were incubated with 50 µl mixed capture beads for 1 h at 25°C. Then, 50 µl phycoerythrin-conjugated detection antibodies were added for 2 h protected from light to form the sandwich complexes. After washing the samples to remove the unbound reagents, the concentrations of the cytokines were determined using a flow cytometer (FACS Array; BD Biosciences, San Jose, USA) and the obtained data were analyzed using the FCAP Array software (BD Biosciences, San Jose, CA, USA).