Fig. 1 Transfection efficiency and fluorescence intensity of K562-GFP cells A: The transfection of K562-GFP cells with GFP was observed by integrating the white light image with the fluorescence image. B: The green fluorescence intensity of K562-GFP cells was analyzed by flow cytometry

Fig. 2 Proliferation curves of K562-GFP cells with different inoculation densities within 48 h monitored in real-time

Fig. 3 Relationship between total intensity and number of cells There is a good linear relationship between the number of cells and the total fluorescence intensity at both the early stage（solid black line）and the growth stage（solid blue line）

Fig. 4 Green fluorescence images of NK cells and K562-GFP cells at different co-culture moments Fluorescence images of different culture groups（columns）, including the single culture group of K562 and the co-culture groups of NK and K562 at different efficiency target ratios（0.1, 0.3, 1, 3, and 9）at different co-culture times（rows）, and co-culture time parameters captured included 0, 4, 8, 24, and 38 h

Fig. 5 Changing curves of NK cells-mediated cytotoxicity leading to K562-GFP cell lysis The proliferation curve of K562-GFP alone（dotted line）and the cytotoxic cleavage curve of K562-GFP cells mediated by NK cells under different effect target ratios（9∶1, 3∶1, 1∶1, 0.3∶1, and 0.1∶1）are monitored in real time within 40 h（solid line）

Fig. 6 Cytotoxicity mediated by NK cells to K562-GFP cells at different co-culture time points The 4PL curves corresponding to different effector target ratios（9∶1, 3∶1, 1∶1, 0.3∶1, 0.1∶1）obtained every 0.5 h during co-culture of effector cells and target cells, where the solid blue line is the 4PL curve of 24 h

Fig. 7 EC50 of NK cells to K562-GFP cells in co-culture stage The real-time change curve of EC50 generated every 0.5 h during co-culture of effector cells and target cells（solid red line）and the results of EC50 coculture for 24 h

Fig. 8 Comparison of two different methods of calculating cytotoxicity A: Cytotoxicity of NK cells at co-culture for 2 h. B: Cytotoxicity of NK cells at co-culture for 4 h. C: Cytotoxicity of NK cells at co-culture for 6 h. D: Cytotoxicity of NK cells at co-culture for 24 h. Late apoptosis indicator 7AAD+ cell population for calculating the cytotoxicity of NK cells to K562-GFP cells（black）, and GFP+ cell population for calculating the cytotoxicity of NK cells to K562-GFP cells（red）

Fig. 9 Correlation between late apoptotic cells and green fluorescent protein degradation The horizontal coordinate is 7AAD+ cell population to calculate the cytotoxicity of NK cells to K562-GFP cells, and the vertical coordinate is GFP+ cell population to calculate the cytotoxicity of NK cells to K562-GFP cells