2.3. Coculture of 3T3-L1 Adipocytes and RAW264.7 Macrophages

Mouse 3T3-L1 pre-adipocytes were co-cultured with the mouse macrophage cell line RAW264.7 as described elsewhere [29]. Briefly, macrophages were seeded on the transwell insert over the previously differentiated 3T3-L1 cells (1 x 10⁵ cells) on day 9 of post-differentiation at a density of 5 x 10⁴ cells, maintaining the cultures at 37 ºC in 5% CO₂, changing the post-differentiation medium every 24 h. After co-culturing for 72 h, medium was replaced by post-differentiation medium without insulin, containing different DIM concentrations for 24 h. At the end of this period, insulin was added at a concentration of 100 nM for 20 min.