Cd3 pc7

Blood samples were taken from healthy adult volunteers who have given informed consent for their blood to be used in this study. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on Ficoll density gradient 1.077 g/cm³ (Paneco, Russia). The NK cells were then magnetically separated using a human NK cell negative selection kit (Miltenyi Biotec, Germany). The percentage of CD3⁻CD56⁺ cells in the preparations after separation was no less than 97% as verified by flow cytometry. In some cases, NK subpopulations were isolated from magnetically separated NK cells by fluorescent-activated cell sorting after staining with monoclonal antibodies CD3-PC7, CD56-APC (Beckman Coulter, USA), and CD57-FITC (eBioscience, USA). Isolated NK cells were cultivated in RPMI-1640 (Paneco) supplemented with 10% FCS (HyClone, USA) (200 μl) at cell concentration of 1.5·10⁶ cells/ml or 0.5·10⁶ cells/ml (for sorted cells) for 18 h in 96 U-well plates (Costar, USA). Recombinant human IL-2 (500 U/ml) purchased from Hoffmann-La-Roche (Germany) and LPS (5 μg/ml) from E. coli strain 055:B5 (Sigma-Aldrich, USA) were added to the cell culture. After incubation, TLR4 and CD69 expression and cytotoxicity of NK cells were analyzed; cell-free supernatants were collected for estimation of cytokine production.