3T3-L1 cells (Health Science Research Resources Bank, Osaka, Japan) were maintained in DMEM supplemented were used for each group. Data are the mean ± SE. *P < 0.05 for STD vs HFD. § P < 0.05 for STD vs HFD + Nar. # P < 0.05 for HFD vs HFD + Nar. STD standard diet, HFD high-fat diet, Nar naringenin ◂ with 10% bovine serum. After pre-adipocytes reached confluence in 12-well plates, they were induced to differentiate into mature adipocytes by replacing medium with 10% FBSsupplemented DMEM containing 0.5 mM IBMX, 0.25 μM DEX, and 5 μg/mL insulin for 2 days. Medium was then replaced with 10% FBS-supplemented DMEM containing 5 μg/mL insulin, and this was changed every 2-3 days for the next 6-7 days.
RAW264 cells (RIKEN BioResource Center, Tsukuba, Japan) were cultured in DMEM supplemented with 10% FBS. Adipocytes and macrophages were co-cultured in a contact system, as described previously (5) . Briefly,
RAW264 cells (1 × 10 5 cells/mL) were plated onto dishes with differentiated 3T3-L1 adipocytes. For each experiment, we treated cells with naringenin and used 0.5% DMSO as the vehicle control. The total volumes of culture media used for the single cell line and the co-culture system treated with naringenin or vehicle control were 0.6 mL and 1.0 mL, respectively.
Tsuhako R., Yoshida H., Sugita C, & Kurokawa M. (2020). Naringenin suppresses neutrophil infiltration into adipose tissue in high-fat diet-induced obese mice. Journal of natural medicines, 74(1).
