Quantifying Secreted IL-1β in Cells

Cells were seeded at 3 × 10⁵ cells per well in a 48-well culture plate (Corning, New York, NY, USA) and grown in serum-free medium overnight. The next day, cells were pretreated with CA and RA alone or in combination with indicated concentrations for 8 h. The supernatants were collected and subjected to centrifugation at 10,000× g for 10 min at 4 °C. The secreted IL-1β in the supernatant was then quantified using an enzyme-linked immunosorbent assay (ELISA) kit for human IL-1β (Raybiotech, Norcross, GA, USA) following the manufacturer’s protocol [18 (link)]. Concentrations of measured IL-1β were normalized to the cell number determined in parallel.