After washing, PBMCs were stained with anti-human antibodies specific for CD2 (PE, RPA-2.10, T-cell marker), CD14 (APC, M5E2, monocyte subset differentiation), CD15 (PE, HIM1, granulocyte marker), CD16 (PE-Cy7, 3G8, monocyte subset differentiation), CD19 (PE, HIB19, B-cell marker), CD56 (PE, MY31, NK-cell marker), CD335 (PE, 9E2, NK-cell marker), HLA-DR (FITC, TU36, antigen-presenting cells) (all from BD Biosciences), as reported by Cros et al., 2010 (link). Cells were acquired on a FACS LSR II flow cytometer (BD Biosciences) and analysed using FlowJo software version 10 (Treestar Inc). For sorting of monocyte subsets, PBMCs were stained and sorted on a MoFlo Astrios cell sorter (Beckman Coulter). Cells were sorted in 1 ml of Isol-RNA lysis reagent (5-Prime GmbH) and frozen at –80°C. To avoid gender-specific effects, three representative male samples of each BioNRW diagnostic group (sCAD, MI, and control) were selected to be subjected to cell sorting and subsequent RNA isolation.
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