图1 K562-GFP细胞的转染效率及荧光强度 A：将K562-GFP细胞的白光图片与荧光图片整合，能够观察到GFP转染K562的情况；B：通过流式分析K562-GFP细胞的绿色荧光强度

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

图2 实时监测不同接种密度的K562-GFP细胞在48 h内的增殖曲线

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

图3 总荧光强度与细胞数量的关系 在早期阶段（黑色实线）和生长期阶段（蓝色实线）两个时间点的细胞数量与总荧光强度均呈现出较好的线性关系

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）

图4 NK细胞与K562-GFP细胞在不同共培养时刻的绿色荧光图片 不同的培养组别（列），包括K562单独的培养组及NK与K562在不同效靶比（0.1、0.3、1、3、9）下的共培养组在不同的共培养时间（行）的荧光图片，拍摄的共培养时间参数包括0、4、8、24、38 h

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

图5 NK细胞介导的细胞毒性导致K562-GFP细胞裂解的变化曲线 实时监测40 h内，单独K562-GFP的增殖曲线（虚线），以及不同效靶比（9∶1、3∶1、1∶1、0.3∶1、0.1∶1）下NK细胞所介导的细胞毒性裂解K562-GFP细胞的变化曲线（实线）

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）

图6 NK细胞在不同共培养时间点对K562-GFP细胞介导的细胞毒性 效应细胞与靶细胞共培养过程中每0.5 h所获取的不同效靶比（9∶1、3∶1、1∶1、0.3∶1、0.1∶1）对应的细胞毒性所生成的4PL曲线，其中蓝色实线为24 h的4PL曲线

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

图7 共培养阶段NK细胞对K562-GFP细胞的EC50 效应细胞与靶细胞共培养过程中每0.5 h所生成的EC50实时变化曲线（红色实线）以及共培养24 h的EC50的结果

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

图8 两种不同细胞毒性计算方式的比较 A：共培养2 h的NK细胞毒性；B：共培养4 h的NK细胞毒性；C：共培养6 h的NK细胞毒性；D：共培养24 h的NK细胞毒性。晚期凋亡指示剂7AAD+细胞群计算NK细胞对K562-GFP细胞的细胞毒性（黑色），GFP+细胞群计算NK细胞对K562-GFP细胞的细胞毒性（红色）

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）

图9 晚期凋亡细胞与绿色荧光蛋白降解的相关性 横坐标为晚期凋亡指示剂7AAD+细胞群计算NK细胞对K562-GFP细胞的细胞毒性，纵坐标为GFP+细胞群计算NK细胞对K562-GFP细胞的细胞毒性

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