2.5. Multiplex Bead-Based Cytokine Assay

After RAW 264.7 cells were seeded in wells of a 96-well plate, poly I:C and/or baicalein were added to the culture medium, and incubation was continued for 24 h at 37 °C. The supernatant was collected from each well containing 10,000 cells, and cytokines were measured using a Luminex assay based on xMAP technology. This assay was performed with Milliplex kits (Millipore, Billerica, MA, USA) and Bio-Plex 200 suspension array system (Bio-Rad) as described previously [8,9,10,11]. The production of the following cytokines was analyzed: IL-1α, IL-6, G-CSF, GM-CSF, VEGF, IP-10, LIX, MCP-1, RANTES, and tumor necrosis factor-alpha (TNF-α). Indomethacin was used as a positive control. Standard curves for each cytokine were generated using the kit-supplied reference cytokine samples.