For several decades, cell‐mediated cytotoxicity has been measured using the ⁵¹Cr release assay. This assay, however, has several drawbacks and flow cytometry has been used as an alternative to measure cytotoxic activity. Here, we present a quantitative method for cell‐mediated cytotoxicity studies, preserving cellular function with minimal sample manipulation. Cytotoxic activity is simply and reproducibly measured as the ability of cytotoxic cells to lyse K562 target cells previously loaded with Calcein‐AM vital stain. After spiking a known number of fluorescent viable K562 target cells into whole blood, cell mixtures are incubated for 2 h in a cell incubator and the remaining spiked cells are counted by flow cytometry. In order to discriminate nucleated cells, erythrocytes, and debris, unlysed whole blood is stained with a cell permeable DNA vital fluorescent dye. Cell‐mediated lysis is measured by comparing target counts for different effector‐to‐target ratios. Since the cytotoxicity of these dyes is relatively low, this method can be broadly applied to studies of innate immune response to tumors and infections, especially where target‐killing activity might be compromised by small volume samples or low frequency of cytotoxic cells. © 2020 International Society for Advancement of Cytometry

中文翻译：

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一种研究全血样品中细胞毒性活性的新型流式细胞术方法

几十年来，细胞介导的细胞毒性一直使用⁵¹Cr 释放测定。然而，这种测定有几个缺点，流式细胞术已被用作测量细胞毒性活性的替代方法。在这里，我们提出了一种用于细胞介导的细胞毒性研究的定量方法，以最少的样品操作保持细胞功能。细胞毒性活性可以简单且可重复地测量为细胞毒性细胞裂解先前加载有 Calcein-AM 活体染色剂的 K562 靶细胞的能力。在将已知数量的荧光活 K562 靶细胞掺入全血后，将细胞混合物在细胞培养箱中培养 2 小时，并通过流式细胞术对剩余的掺入细胞进行计数。为了区分有核细胞、红细胞和碎片，未裂解的全血用细胞可渗透的 DNA 重要荧光染料染色。细胞介导的裂解是通过比较不同效应物与靶标比率的靶标计数来测量的。由于这些染料的细胞毒性相对较低，因此该方法可广泛应用于对肿瘤和感染的先天免疫反应的研究，尤其是在小体积样本或低频率细胞毒性细胞可能会损害靶向杀伤活性的情况下。© 2020 国际细胞计量学促进会