K562 Cell Apoptosis and NK Cell Cytotoxicity Assays

For K562 cell apoptosis assay, purified human primary NK cells were initially activated with DMSO or Daphnetin for 18 h in 96-well V-plate. The K562 cells were labeled with CTV dye (ThermoFisher Scientific, Waltham, United States) and then added to the pre-activated NK cells for co-culture at a 5:1 ratio for another 6 h. The cells were harvested and then the apoptosis kit was used and the ratio of Annexin V^-7-AAD^-, Annexin V⁺7-AAD^-, and Annexin V⁺7-AAD⁺ K562 cells was determined by flow cytometry (18 (link)).
NK cell cytotoxicity assay against K562 cells was also performed by using a real-time digital bio-imaging system, as described previously (19 (link)). Briefly, purified human primary NK cells were initially activated with DMSO or Daphnetin in the presence of IL-12 for 18 h in a 96-well U plate. The K562 cells were labeled with CellTracker dye (ThermoFisher Scientific, Waltham, United States) and then added to the pre-activated NK cells for co-culture at different ratios. Next, the plate was placed in a cell-imaging multimode reader (Cytation 5, Biotek, VT, United States) and the protocol was set to focus on CellTracker-labeled K562 cells in each well at the indicated time points. The fluorescent area of CellTracker-labeled K562 cells in each well was recorded at 4 and 8 h and further analyzed using Gen5™ software (BioTek, VT, United States).