Primary macrophage isolation
ADHESION
Primary macrophages for use in experimental in vitro assays of participant plasma functionalities were isolated from healthy male volunteers in an overnight fasted state. Peripheral blood mononuclear cells were isolated using Ficoll gradient extraction (Sigma, GE17-1440-03) and then cultured in flasks with Roswell Park Memorial Institute Medium 1640 (RPMI) (Thermo Fisher Scientific, 11875119) and 1x penicillin–streptomycin–glutamine (1xPSG) (Thermo Fisher Scientific, 11875119) for 3 h to induce adhesion. Nonadherent cells were discarded, and adherent cells were placed into RPMI, 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, A3160402), and 1xPSG containing 20 ng/mL of human macrophage colony-stimulating factor (Peprotech, 300-25) for 7 d to induce macrophage differentiation.