Macrophage Response to Extracellular Vesicles and Signaling Inhibitors

Cryopreserved RAW264.7 macrophages purchased from ATCC (Manassas, VA) were cultured in a high-glucose DMEM medium containing 10% fetal bovine serum (FBS). The cells were cultured at 37°C, and the medium was changed every 3 days when the cell confluence was approximately 80%.
The RAW264.7 cells were seeded into 24-well plates at 1 × 10⁵ cells/well, and divided into 6 groups: Control group, LPS group, 2D-sEVs group, 4D-sEVs group, IGFBP2-siRNA-#2 and NSC74859 group. First, the LPS group, 2D-sEVs group, 4D-sEVs group, IGFBP2-siRNA-#2 and NSC74859 group were stimulated with 100 ng/mL lipo-polysaccharide (LPS, Sigma-Aldrich, Waltham, MA, United States) for 12 h, and the Control group contained an equal volume of medium without LPS. Next, the supernatant was removed and washed 3 times with PBS. STAT3 inhibitor, S3I-201(NSC74859, S1155; Shanghai, China) is used as described in the article (Zheng et al., 2019 (link)). NSC74859 group was pre-treated with S3I-201 (10 µM) for 1 h. Dil dye-labeled 2D sEVs (40 μg/well) were added to the 2D-sEVs group, and an equal amount of Dil-labeled 4D sEVs/IGFBP2-siRNA-#2-4D sEVs were added to the 4D-sEVs/NSC74859/IGFBP2-siRNA-#2 group for 24 h. Dil dye was added to the Control and LPS group for 24 h. The superna-tant was collected, and the cells were fixed for immunohistochemical staining.