Generation of Resting Macrophages from THP-1 and RAW 264.7 Cells

THP-1 human monocytes (ATCC TIB-202) were suspended in complete RPMI media and incubated at 37°C/5% CO₂ for 24 h in presence of 50 ng/ml phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich), and then for 48 h in complete RPMI media without any stimulus to generate the resting mφs [21 (link)]. RAW 264.7 murine macrophages (ATCC TIB-71) were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 2 mmol/l glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Corning, Corning NY). THP-1 (human) or RAW 264.7 (murine) mφs were seeded in 6-well (1×10⁶ cells/well), 24-well (5×10⁵ cells/well) or 96-well (1×10⁴ cells/well) plates, or in Nunc Lab-Tek II chamber slides (1×10⁴ cells/well, Thermo Scientific, Waltham MA) and incubated for 2 h to allow the cells to adhere. Serum-free media was added, and macrophages were incubated in triplicate with microparticles (MPs) isolated from human or mouse plasma (10% plasma equivalent) or from media of Tc-infected cells (10% media equivalent). Macrophages (± MPs) were incubated for 1, 12, 24 or 48 h and cells and supernatants were stored at −80°C.