Quantifying NK Cell Cytotoxicity against K562 Cells

K562 cells that do not express the Major Histocompatibility Complex (MHC) class I molecules on the cell surface (missing self) were used as classical targets of NK cells to evaluate their cytotoxic activity as previously described (4 (link)). Briefly, K562 cells were stained with PKH Red Fluorescence Cell Linker kit (Sigma-Aldrich Merck, Darmstadt, Germany) and then cocultured with PBMCs (1:2). After 1 h, they were collected and stained with Annexin V conjugated with fluorescein isothiocyanate (FITC) (Thermo Fisher, Waltham, MA, USA) to quantify early apoptosis by measuring the expression of phosphatidylserine on the cell surface by flow cytometry. BD LSRFortessa X-20 flow cytometer and FACS Diva software were used for data acquisition, and FlowJo_V10 software was used for data analysis.