An update to DNA ladder assay for apoptosis detection.
An update to DNA ladder assay for apoptosis detection.
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DOI：
10.15171/bi.2015.01
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发表时间：
2015
期刊：
影响因子：
--
通讯作者：
Barar J
中科院分区：
文献类型：
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作者：
Rahbar Saadat Y;Saeidi N;Zununi Vahed S;Barzegari A;Barar J
简介：近年来，人们对细胞凋亡（程序性细胞死亡）的研究兴趣日益浓厚，这使得人们对细胞-细胞信号传导、细胞-环境相互作用和药物筛选过程中的分子机制有了更好的理解。这反过来又导致了新的检测方法的出现和更准确的快速和早期检测细胞凋亡的试剂盒的开发。然而，它们的敏感性和可靠性往往受到严格审查。本文介绍了一种快速、改进的DNA梯状凋亡检测方法。 方法：采用NIH-3 T3细胞系。在用凋亡剂500 μM H2 O2处理细胞48小时后，提取DNA。采用改良的DNA梯状条带法检测细胞凋亡。流式细胞术和DAPI染色以验证细胞凋亡。 结果：流式细胞仪检测到H2 O2处理后细胞发生了早期和晚期凋亡。用于显示DNA损伤的DAPI染色和使用1.5%凝胶电泳的DNA梯状分析显示处理的细胞的DNA中的片段化。 结论：本研究旨在改进DNA梯状条带法，使其能高质量地检测哺乳动物细胞凋亡。在我们的策略中，采用一个实用的DNA提取方案，DNA梯状电泳可以作为一种简单/快速的方法用于细胞凋亡检测。这种改进的方法能够以成本有效/及时的方式检测细胞凋亡，而不需要商业试剂盒和特殊设备。
Introduction: A growing interest in apoptosis, programmed cell death, in the last years is observed and leads to better understanding of molecular mechanisms during cell–cell signaling, cell-environment interaction and screening of drugs. This in turn results in emerging of new assays and development of more accurate kits for fast and early detection of apoptosis. However, their sensitivity and reliability have often been scrutinized. Here we introduce a rapid and improved method of DNA ladder apoptosis assay for evaluating apoptosis in mammalian cells. Methods: NIH-3T3 cell line was used in this study. After treatment of cells with apoptotic agent, 500 μM H2O2 at 48 hours, DNA was extracted. Then an update protocol of DNA ladder assay was applied for detection of apoptosis. Flow cytometry and DAPI staining were performed to verify apoptosis. Results: Primary and late apoptosis in the H2O2-treated cells was determined by flow cytometry analysis. DAPI Staining used to show DNA damage and DNA ladder assay using 1.5% gel electrophoresis showed fragmentation in the DNA of treated cells. Conclusion: In this research we aimed to improve DNA ladder assay to the high quality detection of apoptosis in mammalian cells. In our strategy, employing a practical DNA extraction protocol, DNA ladder assay could be applied as an easy/fast method for apoptosis detection. This improved method is able to detect apoptosis in a cost effective/timely manner without need for commercial kits and special equipment.