Cardiac-derived Monocyte Activation by EVs

Cardiac-derived monocytes were fluorescence-activated cell–sorted from LVs, 24 hours after the onset of MI. Fluorescence-activated cell–sorted CD45+/CD11b⁺/Ly6G⁻/Ly6C⁺ monocytes were counted, seeded at a density of 4.5×10⁵ cells per well, and cultured in complete RPMI (Roswell Park Memorial Institute)-1640 medium (1% exosome-depleted serum; 1% β-mercaptoethanol and penicillin streptomycin). Cells were then stimulated for 24 hours with lEVs or sEVs derived from MI mice at a ratio of 10⁵ lEVs per cell or 10³ sEVs per cell to reproduce local concentrations of infiltrating monocytes and those of lEVs and sEVs in the infarcted heart 24 hours postligation. NaCl 0.09% was used as control vehicle. TNFα (tumor necrosis factor alpha), CCL (chemokine ligand) 2, CCL7, IL (interleukin)-4, IL-5, IL-6, IL-10, IL-12, IL-13, and IFNγ (interferon gamma) levels were quantified using ProcartaPlex multiplex immunoassays panels (Affymetrix, eBioscience, France).^{10 (link),11 (link)} Concentrations were calculated from a control standard curve.