2.4. HA Concentration Optimization

The concentration of HA was optimized based on the concentration of cytokines released from THP-1 cells after activation with lipopolysaccharide (LPS). THP-1 cells were seeded in 24-well plates at a density of 450,000 cells per well. Phorbol 12-myristate 13-acetate (50 ng/mL) was used to differentiate THP-1 cells into macrophages. After two days of resting, the cells were exposed to LPS (1 µg/mL) and different concentrations of HA (0.5, 1, 2, 5, and 10 mg/mL) for 24 h. Sterile HA was used in all experiments. The supernatant was collected, and cytokine release (IL-6 and IL-8) was determined using an ELISA kit (Biolegend, San Diego, CA, USA).