Department of Molecular Biotechnology, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey.
Department of Molecular Biology and Genetics, Faculty of Science, Karadeniz Technical University, 61080, Trabzon, Turkey.
J Fluoresc. 2018 Nov;28(6):1393-1404. doi: 10.1007/s10895-018-2306-4. Epub 2018 Oct 21.
The evaluation of cell wellness is an important task for molecular biology research. This mainly comprises the assessment for morphology and viability of culturing cells. Annexin V-Propidium iodide counterstaining has been currently one of the common and easy methods to discriminate apoptotic and necrotic cell profiles. The method is operated by fluorescence-based detection of counterstain via laser beam-employed instruments including flow cytometer, fluorescence microscope and automated cell counter. The detection is primarily conducted based on the same principle; however the efficiency of instruments may vary. Here we evaluated the efficiency of those instruments for the clear-cut detection of cell death through various mammalian and microalgae cell lines. To the best of our knowledge, this is the first study revealing comparative analyses of apoptotic and necrotic cells in mammalian and microalgae cells using Annexin V-PI counterstain detected by flow cytometer, fluorescence microscope and automated cell counter. Fluorescence microscope and cell counter instruments were also tested and compared for the traditional trypan blue-based cell viability detection performance. For these, cell death was induced by UV-irradiation and/or bee venom for mammalian (pancreatic cancer, metastatic breast cancer and mouse fibroblasts) and microalgae cells (Chlorella vulgaris), respectfully. Findings postulated that automated cell counter and fluorescence microscopy revealed similar patterns for the detection by both counterstain and trypan blue in mammalian cells. Interestingly, flow cytometry did provide an accurate and significant detection for only one mammalian cell line when UV-treatment was followed by routine Annexin V-Propidium iodide counterstaining. Unlike, only flow cytometry revealed a significant change in the detection of death of microalgae cells by Annexin V-Propidium iodide method, but both Annexin and conventional trypan blue methods were not applicable for the automated cell counter and microscopic detections for microalgae cells. The related outputs propose that the obtaining reliable quantitation strongly depends on cell type and instruments used. These suggest the necessity of optimization and validation endeavors before any cell death detection initiative. The analytical outcomes present insights into detailed assessment of cell death detection of eukaryotic cells and provide a direction to researchers to consider.
细胞活力评估是分子生物学研究的一项重要任务。这主要包括培养细胞形态和活力的评估。膜联蛋白 V-碘化丙啶复染目前是区分凋亡和坏死细胞形态的常用且简单的方法之一。该方法通过荧光检测来区分细胞死亡类型，使用的仪器包括流式细胞仪、荧光显微镜和自动细胞计数器。这些仪器的检测原理基本相同，但效率可能有所不同。在这里，我们评估了这些仪器在通过各种哺乳动物和微藻细胞系清晰检测细胞死亡方面的效率。据我们所知，这是首次使用流式细胞仪、荧光显微镜和自动细胞计数器检测 Annexin V-PI 复染来分析哺乳动物和微藻细胞中凋亡和坏死细胞的比较分析的研究。我们还测试和比较了荧光显微镜和细胞计数器仪器在传统台盼蓝法检测细胞活力方面的性能。为此，我们通过紫外线照射和/或蜂毒诱导哺乳动物（胰腺癌、转移性乳腺癌和小鼠成纤维细胞）和微藻（普通小球藻）细胞死亡。结果表明，自动细胞计数器和荧光显微镜在检测哺乳动物细胞时，无论是复染还是台盼蓝染色，显示出相似的模式。有趣的是，只有在 UV 处理后常规进行 Annexin V-碘化丙啶复染时，流式细胞术才能准确检测到一种哺乳动物细胞系的凋亡。相比之下，只有流式细胞术能够检测到微藻细胞通过 Annexin V-碘化丙啶方法死亡的显著变化，而 Annexin 和传统台盼蓝方法都不适用于自动细胞计数器和显微镜检测微藻细胞。这些结果表明，获得可靠的定量结果强烈依赖于细胞类型和使用的仪器。这表明在进行任何细胞死亡检测之前，需要进行优化和验证工作。这些分析结果为真核细胞死亡检测提供了详细的评估，并为研究人员提供了一个考虑的方向。
