Ficoll percoll gradients

Peripheral blood mononuclear cells (PBMCs) were prepared from fresh-heparinized venous blood of bovine and ovine by centrifugation over Ficoll-Histopaque (Sigma, St. Louis, MO, United States) as described (Huang et al., 2011 (link)). M/MΦs cells were enriched from PBMCs using Ficoll-Percoll gradients (GE Healthcare) and further purified by anti-CD14 magnetic beads with column purification according to the manufacturer’s instructions (Miltenyi Biotec; purity of cells >95%). The enriched cells were cultured in Roswell Park Memorial Institute-1640 medium supplemented with 10% FBS.

Peripheral blood mononuclear cells (PBMCs) were prepared from healthy adult donors by centrifugation over Ficoll-Histopaque (Sigma, St. Louis, MO, USA) as described (21 (link)). All the donors had signed an informed consent before their blood was used in this study. M/MΦs cells were enriched from PBMCs using Ficoll-Percoll gradients (GE Healthcare) and further purified by anti-CD14 magnetic beads with column purification according to the manufacturer’s instructions (Miltenyi Biotec; purity of cells >95%). The enriched cells were cultured in RPMI 1640 (Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone).
The human monocytic cell line THP-1 and HEK-293 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). THP-1 were maintained in RPMI 1640 medium and HEK-293 cells were maintained in Dulbecco’s minimum essential medium, both supplemented with 10% FBS at 37°C with 5% CO₂.