Xcelligence impedance based rtca system

The cytotoxicity of NK cells was assessed with a real-time cell analysis (RTCA) assay. Adherent target MCF-7 (3 × 10⁴ cells/well), MDA-MB-231 (3 × 10⁴ cells/well), or SK-BR-3 (4 × 10⁴ cells/well) cells were seeded onto 16-well E-Plates (ACEA Biosciences) in 150 μL of standard RPMI-1640 full medium. For nonadherent K562 cells, 16-well E-Plates were precoated with the Liquid Tumor Killing Assay (anti-CD71) Tethering Kit (ACEA Biosciences) according to the manufacturer's recommendations. K562 cells were seeded onto 16-well E-Plates at a cell density of 1.5 × 10⁴ cells/well. The proliferation of target cells was monitored in the incubator at 37°C (5% CO₂, 95% humidity) for 24 hours with the xCELLigence impedance-based RTCA system (Acea Biosciences). The next day, 100 μL of the medium was aspirated and replaced with the medium containing effector cells (human primary NK cells or NK-92 cell lines) at different effector to target (E:T) ratios. For antibody-dependent cell cytotoxicity (ADCC) assays, trastuzumab (anti-HER2 antibody) was added to appropriate wells at a final concentration of 10 μg/mL. The cells were monitored for the next 20 to 24 hours. Analysis was performed using RTCA Software Pro (ACEA Biosciences). The impedance changes (cell index) were normalized to the end value of the target cells' proliferation and plotted over time as normalized cell index.