Jang Youn-Young, Cho Duck, Kim Sang-Ki, Shin Dong-Jun, Park Min-Ho, Lee Je-Jung, Shin Myung-Geun, Shin Jong-Hee, Suh Soon-Pal, Ryang Dong-Wook
Research Center for Cancer Immunotherapy, Chonnam National University Hwasun Hospital, Jeollanamdo, Korea.
Ann Clin Lab Sci. 2012 Winter;42(1):42-9.
Several flow cytometric methods for measuring natural killer cell activity have been developed. Commonly used protocols involve the staining of target cells with various fluorescent dyes. However, these protocols are not applicable to certain experimental settings. Therefore, we used Calcein AM (CAM), which has been reported to be the most suitable dye for use in target cell staining protocols, as a means of developing an improved flow cytometry-based NK cytotoxicity assay involving effector cell staining. Peripheral blood mononuclear cells (PBMCs) isolated by gradient density centrifugation and expanded NK cells were used as effector cells. Cytotoxicity against K562 cells and several hematologic cancer cell lines was measured by a flow cytometry-based method using CAM and propidium iodide. The new assay was compared with a standard (51)Cr release assay (CRA) in terms of its ability to measure the cytotoxicity of NK cells in PBMCs and expanded NK cells against K562 cells. The optimal concentration of CAM for staining effector cells was 0.05 μM, and CAM fluorescence intensity in effector cells was maintained for 4 hours. CAM staining had no significant effect on NK cell activity in human PBMCs or expanded NK cells. Comparison of the CRA and this new assay using K562 cells revealed a good correlation (PBMCs, r = 0.894; expanded NK cells, r = 0.887). Distinct separation between target tumor cells (Daudi, Raji, RPMI8226, U266, U937, and K562 cells) and CAM-stained PBMCs (E:T ratio, 12.5:1 to 50:1) or expanded NK cells (E:T ratio, 0.5 to 4:1) was observed after incubation for 1 or 4 hours. In summary, we successfully developed an effective flow cytometry-based assay for assessing the activity of NK cells in PBMCs and expanded NK cells against K562 cells and various types of hematologic cancer cells.
已经开发了几种用于测量自然杀伤细胞活性的流式细胞术方法。常用的方案包括用各种荧光染料对靶细胞进行染色。然而，这些方案不适用于某些实验设置。因此，我们使用了已被报道为最适合用于靶细胞染色方案的染料——钙黄绿素乙酰甲酯（CAM），作为开发一种改进的基于流式细胞术的NK细胞毒性测定方法的手段，该方法涉及效应细胞染色。通过梯度密度离心分离的外周血单个核细胞（PBMC）和扩增的NK细胞用作效应细胞。使用CAM和碘化丙啶通过基于流式细胞术的方法测量对K562细胞和几种血液系统癌细胞系的细胞毒性。就其测量PBMC和扩增的NK细胞中NK细胞对K562细胞的细胞毒性的能力而言，将新测定法与标准的（51）铬释放测定法（CRA）进行了比较。用于染色效应细胞的CAM的最佳浓度为0.05μM，并且效应细胞中的CAM荧光强度保持4小时。CAM染色对人PBMC或扩增的NK细胞中的NK细胞活性没有显著影响。使用K562细胞对CRA和这种新测定法进行比较显示出良好的相关性（PBMC，r = 0.894；扩增的NK细胞，r = 0.887）。在孵育1或4小时后，观察到靶肿瘤细胞（Daudi、Raji、RPMI8226、U266、U937和K562细胞）与CAM染色的PBMC（效靶比，12.5:1至50:1）或扩增的NK细胞（效靶比，0.5至4:1）之间有明显的分离。总之，我们成功开发了一种有效的基于流式细胞术的测定方法，用于评估PBMC和扩增的NK细胞对K562细胞和各种类型血液系统癌细胞的活性。
