*4.4. Cytokine Assays*

RAW264.7 cells were pre-treated with coelonin for 1 h and then stimulated with LPS (200 ng/mL) for 12 h, 0.05% DMSO was applied as the parallel solvent control. The culture supernatant was collected for IL-6 and TNF-α detection. Then, the remaining cells were treated with 1 mM ATP for an additional 15 min at 37 ◦C [18], and supernatants were collected for IL-1β detection. IL-6, TNF-α and IL-1β levels were quantified using the cytometric beads array (CBA) method, according to the manufacturer's protocols (BD, Franklin Lakes, NJ, USA), performed on a BD Accuri™ C6 flow cytometer (BD, Ann Arbor, MI, USA).
