流式细胞术检测NK细胞的细胞毒作用的两种方法比较

目的:比较并优化流式细胞术检测人NK细胞体外细胞毒功能的方法。方法:采用Calcein-AM释放法或CFSE/7-AAD法分别对靶细胞进行标记,按10∶1和20∶1效靶比与7例NK细胞在37℃5%CO2培养箱共培养4 h,流式细胞术测定两种不同标记方法的靶细胞死亡率。结果:各实验组中CFSE/7-AAD法检出的靶细胞死亡率均高于Calcein-AM释放法,在低效靶比、弱细胞毒作用条件下,差异具有统计学意义。细胞毒作用较弱时,Calcein-AM释放法平均荧强度变化不显著、结果误差较大;而CFSE/7-AAD法则能更加敏感地检出靶细胞的死亡率。结论:CFSE/7-AAD法能够特异、灵敏地检出NK细胞的细胞毒活性,所获得的结果更为可靠。

A comparison of NK cell-mediated cytotoxicity detected by flow cytometry using two different staining methods

Objective:To compare and optimize a method for detecting cytotoxicity mediated by vitro expanded NK cells using flow cytometry.Methods:High purity NK cells were applied as effector cells.Calcein-AM release or CFSE/7-AAD was used to label target cells respectively.Seven different NK cells and different target cells were co-incubated at 37℃ 5% CO2 incubator for 4 hours with a E∶T ratio of 10∶1 or 20∶1,respectively.The death rate of target cells was detected by flow cytometry.Results:Comparing the two different staining method,CFSE/7-AAD double staining method showed better capability than Calcein-AM release in detecting death rate of target cells,particularly in the condition of lower E∶T ratios and weak cytotoxicity.Under situation of weak cytotoxicity,the mean fluorescence intensity(MFI)of target cells labeled by Calcein-AM release did not show significant alteration and may result in measurement error in calculating the death rate of target cells.Conclusion:CFSE/7-AAD double staining method exhibits more sensitive in detecting cytotoxicity mediated by NK cells and the obtained results are more reliable.