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首页> 外文期刊>Protoplasma: An International Journal of Cell Biology >Analysis of cytotoxicity of melittin on adherent culture of human endothelial cells reveals advantage of fluorescence microscopy over flow cytometry and haemocytometer assay
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Analysis of cytotoxicity of melittin on adherent culture of human endothelial cells reveals advantage of fluorescence microscopy over flow cytometry and haemocytometer assay

机译：蜂毒肽对人内皮细胞粘附培养的细胞毒性分析显示，荧光显微镜优于流式细胞仪和血细胞计数器

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Melittin, from the honeybee venom, is a membrane active protein, whose cytotoxicity to human endothelial cells has not been described yet. In this work, we studied its time-dependent cytotoxicity on human umbilical vein endothelial cells (HUVECs). Since HUVECs grow in culture as adherent cells, suspension of cells is required before measuring cytotoxicity with a haemocytometer or flow cytometry. Therefore, we also tried to discover whether the result of cytotoxicity tests of melittin is influenced by the preparation of the cell suspension. For this purpose, we compared the results of haemocytometer-based trypan blue assay and flow cytometry using 7-aminoactinomycin D (7-AAD) with results of fluorescence microscopy using 7-AAD and 4', 6-diamidino-2-phenylindole (DAPI). Melittin over 60 min exposure evoked a rapid decline in the survival of HUVEC. After 60 min exposure to melittin, the phase contrast microscopy demonstrated massive necrosis in the remaining attached cells. Fluorescence microscopy detected both viable and non-viable cells in adequate proportions at all exposure times, whereas haemocytometer-based assay and flow cytometry highly underestimated the percentage of non-viable cells or even failed to detect any dead cells. Our data clearly indicate that the induction of large-scale damage to adherent endothelial cells by melittin results in a loss of the majority of necrotic cells during sample preparation for flow cytometry or a haemocytometer-based assay. In the case of adherent cell culture, therefore, fluorescence microscopy was shown to be a more appropriate method for quantitative analysis of cell death caused by a fast-acting cytolytic toxin such as melittin.
机译：来自蜜蜂毒液的蜂毒肽是一种膜活性蛋白，其对人内皮细胞的细胞毒性尚未见报道。在这项工作中，我们研究了其对人脐静脉内皮细胞（HUVEC）的时间依赖性细胞毒性。由于HUVEC在培养时会以贴壁细胞的形式生长，因此在使用血细胞计数器或流式细胞仪测量细胞毒性之前，需要先悬浮细胞。因此，我们还试图发现蜂毒蛋白的细胞毒性测试结果是否受到细胞悬液制备的影响。为此，我们将使用7-氨基放线菌素D（7-AAD）的基于血细胞计数器的锥虫蓝测定和流式细胞术的结果与使用7-AAD和4'，6-diamidino-2-phenylindole（DAPI）的荧光显微镜的结果进行了比较）。蜂毒素在60分钟以上的暴露中引起HUVEC生存迅速下降。暴露于蜂毒肽60分钟后，相差显微镜显示剩余的附着细胞中大量坏死。荧光显微镜在所有暴露时间都以适当的比例检测到了活细胞和非活细胞，而基于血细胞计数器的分析和流式细胞术则大大低估了非活细胞的百分比，甚至未能检测到任何死细胞。我们的数据清楚地表明，蜂毒素引起的对贴壁内皮细胞的大规模破坏导致在流式细胞仪或基于血细胞计数器的样品制备过程中大多数坏死细胞的丢失。因此，在贴壁细胞培养的情况下，荧光显微镜被证明是定量分析由速效溶细胞毒素（如蜂毒肽）引起的细胞死亡的更合适方法。

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