To investigate the role of AT-EVs in inflammation, 5 × 105 RAW 264.7 macrophages were seeded in six-well plates and cultured with high-glucose DMEM with FBS, until cells reached 80% confluence. As previously described (Xu et al., 2020 (link)), cells were divided into five groups for which the culture medium was replaced as follows: Control group : cells were cultured with DMEM; Lipopolysaccharide (LPS) + IFNγ group : cells were cultured with DMEM +1 μg/mL LPS (#L2880, Sigma-Aldrich, St. Louis, MO, United States) + 30 ng/mL IFNγ (#315-05-100, PeproTech, Rocky Hill, NJ, United States); LPS + IFNγ + dAT-EVs group : cells were cultured with 1 μg/mL LPS +30 ng/mL IFNγ +150 μg/mL dAT-EVs; LPS + IFNγ + mAT-EVs group : cells were cultured with 1 μg/mL LPS +30 ng/mL IFNγ +150 μg/mL mAT-EVs; LPS + IFNγ + cAT-EVs group : cells were cultured with 1 μg/mL LPS +30 ng/mL IFNγ +150 μg/mL c AT-EVs. After induction for 24 h, 2 × 105 cells were incubated with fluorescein isothiocyanate (FITC)-anti-mouse CD86 (1:50, #105110, BioLegend, San Diego, CA, United States) and PE-anti-mouse CD206 (1:40, #141706, BioLegend) at 4°C for 30 min, before being washed twice with staining buffer (#00-4222-26, Invitrogen, San Diego, CA, United States of America). Polarization of RAW 264.7 cells was determined using flow cytometry (BD FACSCalibur, Beckman Coulter, Inc., Brea, CA, United States).
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