PBMCs were isolated by Ficoll-Paque Plus (Cytiva Europe GmbH) density gradient centrifugation and then washed twice in culture medium (RPMI-1640 supplemented with 10% FBS) [30 (link)]. T cell isolation was performed through immunomagnetic cell sorting using CD4/CD8 microbeads (MACS Miltenyi Biotec). Human monocytes isolation was performed through immunomagnetic cell sorting using CD14 microbeads (MACS Miltenyi, Biotec) according to the manufacturer instructions.
For macrophage differentiation CD14+ monocytes (1 × 106/ml) from healthy PBMCs were seeded in RPMI-1640 medium, 100 ng/ml M-CSF (Miltenyi, Biotec) for 7 days in 5% CO2 at 37 °C. On day 8, the medium was replaced with fresh RPMI-1640 and macrophages were plated with EOC cell lines.
For macrophage differentiation CD14+ monocytes (1 × 106/ml) from healthy PBMCs were seeded in RPMI-1640 medium, 100 ng/ml M-CSF (Miltenyi, Biotec) for 7 days in 5% CO2 at 37 °C. On day 8, the medium was replaced with fresh RPMI-1640 and macrophages were plated with EOC cell lines.
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