ELISA measurement of IL-6, IL-1β, TNF-α, and IFN-γ in primary microglia and ACC tissue

Primary microglia and ACC samples underwent homogenization in physiological saline via an automated rapid sample homogenizer, followed by centrifugation at 1,000g for 10 min in a low-temperature high-speed centrifuge maintained at −4 °C. The processed samples were then aliquoted and preserved at −80 °C for future examination.

For cytokine quantification, we employed mouse IL-6, IL-1β, TNF-α, and IFN-γ Valukine ELISA Kits from BioTechne (USA), adhering strictly to the manufacturer’s guidelines. The cytokine levels were determined using an EnSpire Multimode Plate Reader provided by PerkinElmer (USA).