This was followed by incubation with labelled polymer alkaline phosphatase (included
in the kit) and a 10-min incubation with Permanent red substrate-chromogen. In some samples, APAF-1 and GNLY were labelled using peroxidase and alkaline phosphate staining, respectively. Interleukin-15 and MHC class I molecules were single labelled using the mouse IgG1 anti-IL-15 Lumacaftor mAb (clone 34593; 1:100 dilution; R&D Systems, Minneapolis, MN, USA) or mouse IgG1 anti-MHC class I mAb (clone W6/32 Department of Physiology and Immunology, Medical Faculty, University of Rijeka, Croatia) and alkaline phosphate staining. Nuclei were stained with Shandon haematoxylin solution (Termo Scientific, Soeberg, Denmark), and the specimens were mounted using Acquatex (MerckKGa, Darmstadt, Germany). Cytospins were single labelled for GNLY using the same kit and the above-described protocol. Slides were analysed with an Olympus B × 51 microscope using an Olympus DP71 camera (Olympus,
Tokyo, Japan). Images were processed using Cell^F imaging software or Cell^A imaging software, version 3.0 (both from Olympus, Tokyo, Japan) and Adobe Photoshop, version 7.0.1 CE (Adobe Systems Incorporated, San Jose, CA, USA). Cytotoxicity assay. NK cell-mediated cytotoxicity was analysed against the NK-sensitive human erythroleukaemia cell line K562 (provided by
Proteases inhibitor Prof. E. R. Podack, Department of Immunology and Microbiology, School of Medicine, University of Miami, Florida, USA) using the method described previously . K562 target cells were labelled with PKH26 lipophilic dye following the manufacturer’s instructions (PKH26 Red Fluorescent Cell Linker Kit; Sigma Biosciences, St. Louis, MO, USA) prior to set up with peripheral blood lymphocytes (PBL) at effector to target cell ratios of 6:1, 12.5:1, 25:1 and 50:1. Samples of PBL and K562 cells cultured in the medium alone served as controls. The samples were incubated O-methylated flavonoid for 18 h at 37 °C in a humidified atmosphere containing 5% CO2. After incubation, samples were labelled with FITC-conjugated annexin V (BD Pharmingen, San Diego, CA, USA) following the manufacturer’s instructions (5 μg/105 cells, for 15 min at room temperature in the dark). Propidium iodide (PI, Sigma-Aldrich Chemie) at a final concentration of 5 μg/mL was added before analysis using FACSCaliburTM. Some PBL samples were pretreated with anti-perforin δG9 mAb (10 μg/105 PBL, provided by Prof. E.R. Podack), anti-GNLY RC8 mAb (10 μg/105 PBL) or both anti-perforin mAb and anti-GNLY mAb at the indicated concentrations.