The flow cytometry-based assay has been increasingly used to assess the cell-mediated cytotoxicity since the 1980s due to its advantages over the conventional radioactive ⁵¹Cr release assay (CRA), such as higher sensitivity at the single-cell level and nonradioactivity. The basic principle of this assay is the usage of two dyes, one nontoxic dye for labeling targets or effector cells to distinguish one from another, one viability dye for discrimination of dead from live cells. Due to the problem of spontaneous release or leakage of the nontoxic dye, the concern about the cross-staining has not yet been clearly elucidated. In this study, carboxyfluorescein diacetate succinimidyl ester (CFSE) was utilized to label target cells and Hoechst 33258 was used as the viability dye. We confirmed that no cross-staining occurred between the effector and target cells after 4 h of coculture. We also found that the cytotoxicity would be overestimated if effector cells instead of target cells were labeled due to the exclusion of viable targets in effector-target conjugates. Using EDTA at the end of culture or labeling targets can solve this problem. Furthermore, the gating strategy could be improved by plotting CFSE against forward scatter (FSC) to discriminate some early apoptotic events. Due to the loss of target cells lysed by effector cells, counting beads are normally preferable in this assay. Here, we found an alternative to the use of beads in standardizing the flow cytometry-based assay. Instead of using beads, sample acquisition in a fixed time was shown to have the same effect in specific lysis evaluation as the beads application but have a greater stability than the latter. With a good quality control, the acquisition time for each sample could be shortened to 15 s, thus making this work to be done efficiently, especially in the case of larger sample sizes. Collectively, the findings in this study can improve the flow cytometric cytotoxicity assay to be carried out in a more accurate, efficient, and cost-effective way. © 2020 International Society for Advancement of Cytometry

中文翻译：

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基于流式细胞术的细胞毒性测定的改进

自 1980 年代以来，基于流式细胞术的分析越来越多地用于评估细胞介导的细胞毒性，因为它优于传统的放射性⁵¹Cr 释放测定 (CRA)，例如在单细胞水平和非放射性方面具有更高的灵敏度。该测定的基本原理是使用两种染料，一种用于标记靶标或效应细胞的无毒染料以区分另一个，另一种用于区分死细胞和活细胞的活力染料。由于无毒染料的自发释放或泄漏问题，对交叉染色的担忧尚未明确阐明。在本研究中，羧基荧光素二乙酸琥珀酰亚胺酯 (CFSE) 用于标记靶细胞，Hoechst 33258 用作活性染料。我们确认在共培养 4 小时后，效应细胞和靶细胞之间没有发生交叉染色。我们还发现，如果效应细胞而不是靶细胞被标记，则细胞毒性会被高估，因为效应细胞-靶标偶联物中排除了活靶标。在培养结束或标记目标时使用 EDTA 可以解决这个问题。此外，可以通过绘制 CFSE 与前向散射 (FSC) 来区分一些早期凋亡事件来改进门控策略。由于效应细胞裂解的靶细胞丢失，因此在该测定中通常优选使用计数珠。在这里，我们发现了在标准化基于流式细胞术的测定中使用珠子的替代方法。与使用珠子不同，固定时间的样品采集在特定裂解评估中与珠子应用具有相同的效果，但比后者具有更高的稳定性。凭借良好的质量控制，每个样本的采集时间可以缩短到 15 秒，从而使这项工作高效完成，尤其是在样本量较大的情况下。总的来说，这项研究的发现可以改进流式细胞仪细胞毒性测定，以更准确、更有效和更具成本效益的方式进行。© 2020 国际细胞计量学促进会