Ultra low attachment 24 well flat bottom plates

Monocyte-derived macrophages were generated from buffy coats (Blood Transfusion Service, Sheffield, UK). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using Histopaque-1077 (Sigma-Aldrich, UK). The PBMC layer was collected and washed, and the monocyte fraction (CD14⁺ cells) was obtained by positive selection using human CD14 MicroBeads and LS MACS columns (Miltenyi Biotec, UK), according to the manufacturer’s instructions. Purified monocytes were suspended in RPMI complete medium (RPMI 1640 [Sigma-Aldrich, UK] containing 15% human AB serum [PAA Laboratories Ltd., UK], 2 mM l-glutamine [Sigma-Aldrich, UK], and 10 mM HEPES [Invitrogen, UK]) and 10 ng/ml recombinant human GM-CSF (rhGM-CSF) (premium grade; Miltenyi Biotec, UK) and plated on ultra-low-attachment 24-well flat-bottom plates (Corning Incorporated, USA) in 500 μl RPMI complete medium containing rhGM-CSF at a density of 1.5 × 10⁶ monocytes/ml. On day 3, 500 µl per well of fresh RPMI complete medium containing rhGM-CSF was added. Macrophages were collected at day 7, washed 3 times in serum-free RPMI 1640, counted, and adjusted to densities appropriate for each assay.