Figure 2.

In Vitro Macrophage Assay Design

Schema showing the different steps involved in the in vitro macrophage assay. First, macrophages (∼2 × 10⁶ cells) were recovered from a frozen vial in 25 mL medium supplemented with 10% FBS and antibiotics. After 24 h of incubation at 37°C in 5% CO₂, the cells were detached by gentle scraping with a cell scraper. Cells were counted and seeded in triplicate at a density of 1 × 10⁵ cells per square centimeter in a volume of 300 μL DMEM containing 5% FBS plus antibiotics. The stimulation was performed by replacing the 300 μL culturing medium with a similar volume of medium alone (5% FBS and antibiotics) or containing LPS (10 ng/mL), LPS plus dexamethasone (1 μg/mL), or LPS plus exosomes (0.5 × 10⁹ vesicles per milliliter). The supernatant was collected after 4 h of incubation at 37°C and kept frozen until interleukin determination by ELISA.