RAW264.7 cells were treated with LPS (1 μg/mL) or different concentrations (15.6–125 μg/mL) of HMF. After incubation for 24 h, RAW264.7 cells were collected and blocked with PBS containing 5% BSA for 15 min. Flow cytometry buffer (PBS containing 1% BSA) was then added to stop blocking. RAW264.7 cells were stained with PE-conjugated anti-CD40 antibody. Furthermore, RAW264.7 cells were stained with APC-conjugated anti-mouse CD86 antibody or APC-conjugated anti-mouse CD206 antibody to detect M1- or M2-polarized macrophages, respectively, using Aria FACS flow cytometry system (Beckman Coulter MoFlo XDP, Fullerton, CA, USA).
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