Cd335

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.

Surface marker staining of PBMCs from patients’ fresh blood samples for the following markers was performed in PBS at 4°C for 30 min following Fc blockade: CD3 (BD, UCHT1), CD56 (BD, NKCM16), CD335 (NKp46) (BD, 9E2/NKp46), CD337 (NKp30) (BD, p30-15), CD57 (BD, NK-1), DNAM1 (BD, DX11), NKG2C (CD159c) (Miltenyi Biotec, REA205), NKG2A (CD159a) (Miltenyi Biotec, REA110), NKG2D (BD, 1D11), KIR2DL1 (BD, HP-3E4), KIR2DL2/L3 (Beckman Coulter), KIR3DL1 (Miltenyi Biotec, REA168), CD107a (BD, H4A3), and IFN-γ (BioLegend, B27). Acquisition was performed on an LSR Fortessa instrument (BD Biosciences), and Flowjo software (TreeStar) was used for analysis. Single-stain controls for compensation were generated utilizing UltraComp eBeads (eBioscience, BioLegend, and Miltenyi Biotec), and the analysis of acquired samples was performed in FlowJo (FlowJo, LLC, Ashland, CA) data analysis software. Data were gated using FlowJo (FlowJo, LLC). Following data acquisition, the channel intensity was normalized using calibration beads. Before the experiment, we verified the configuration by selecting an appropriate configuration and defining a new baseline every 24 h. We set up and ran a performance check. The anti-human monoclonal antibodies used for flow cytometry are listed in Supplementary Table 1.