Primary human monocyte and macrophage culture

With informed consent and ethics approval from the Human Research Ethics Committee of the Royal Prince Alfred Hospital (protocol number X16-0300), whole blood samples were obtained from three healthy male individuals (Donors N1, N2 and N3). Blood samples were diluted in Hanks’ balanced salt solution (HBBS) in a ratio of 1:2. Following addition of Ficoll and gradient separation, peripheral blood mononuclear cells (PBMCs) were isolated and washed. PBMCs were then frozen in cryopreservation medium containing RPMI medium supplemented with 40% (v/v) FBS and 10% (v/v) DMSO and stored in liquid nitrogen. For CD14⁺ cell selection, PBMCs were thawed and underwent magnetic-activated cell sorting (MACS) separation according to the manufacturers’ instructions (Miltenyi Biotec). Briefly, cells were stained with anti-CD14 phycoerythrin (PE, BioLegend) and anti-PE MicroBeads. Cells were then placed into LS columns and underwent MACS separation. Labelled CD14⁺ monocytes were collected following washing in phosphate-buffered saline.

Purified monocytes were maintained in RPMI medium supplemented with 2 mM L-glutamine, 25 mM HEPES, 20% (v/v) FBS, and 0.1 mg/ml penicillin/streptomycin at 37°C in the presence of 5% CO₂. Monocytes were differentiated into macrophages using 100 ng/ml M-CSF (PeproTech) for 7 days, with culture media being replaced every 3 days. Following differentiation, macrophages were further polarised to a proinflammatory phenotype using 100 ng/ml LPS and 50 ng/ml IFN-γ for 8 h.