2.7. Macrophage Activation
BMDMs (105 cells/well) were incubated with synthetic peptides and 100 μg/mL LDL (−) or PBS as negative control and 10 μg/mL LPS as positive control for 3 h at 37 °C. Total RNA was extracted with TRIzol (Life Technologies, Carlsbad, CA, USA) following the manufacturer’s instructions. The extracted RNA was quantified by spectrophotometry. cDNA was constructed from RNA samples by RT-PCR with Superscript Vilo (Life Technologies, Carlsbad, CA, USA). qPCR was performed using the 7500 Fast Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) to analyze the expression levels of NOS2, COX-2, TNF-α, IL-1α, TGF-β, and IL-10. Activation of macrophages by LPS was evaluated as a positive control (Supplementary Materials (C. Macrophage characterization; Figure S7)). To verify if BMDM activation was dependent on the endocytosis of peptides an experiment was carried out with Brefeldin A. For this 106 BMDM/well were treated for 1 h with 1 μM Brefeldin A. Then, the cells were washed with PBS and incubated with 100 μg/mL P2C7 in RPMI 1640, 10% FBS and Brefeldin A (1 μM) for 3 h. A negative control was done only with RPMI 1640, 10% FBS and Brefeldin A (1 μM) incubated for 3 h. Further the expression of NOS2, TNF-α and TGF-β was evaluated by qPCR assay.