4.7. NF-κB Activation Assay

A549 cells were transfected with a NF-κB reporter plasmid, pNF–κB–Luc (A549–NF-κB–Luc cells) using BioT transfection kit (Bioland Scientific, Paramount, CA, USA). A549–NF-κB–Luc cells were grown to 70% confluence in a 96-well plate. Viral particles (MOI 1) were incubated with 20 µg/mL of C1q, ghA, ghB, ghC, or MBP for 1 h at room temperature and then for another h at 4 °C. The pre-treated viral particles were used to challenge the A549–NF-κB–Luc cells in infection media for 6 h. Virus particles that were not treated with C1q, or treated with MBP alone were used as control. A549–NF-κB–Luc cells treated with recombinant TNF-α (10 ng/ml) and IL-1β (10 ng/ml) (R&D Systems) were used as positive controls for NF-κB activation. Luminescence proportional to the NF-κB activity was measured in relative units using a Clariostar Plus Microplate Reader (BMG Labtech, Ortenberg, Germany).