A simple technique for quantifying apoptosis in 96-well plates
A simple technique for quantifying apoptosis in 96-well plates
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DOI：
10.1186/1472-6750-5-12
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发表时间：
2005-05-10
影响因子：
3.5
通讯作者：
Shellman, YG
中科院分区：
文献类型：
--
作者：
Ribble, D;Goldstein, NB;Shellman, YG
背景：分析细胞凋亡已成为许多生物学研究的组成部分。然而，目前可用的用于定量凋亡的方法具有各种限制，包括多个，有时是细胞损伤步骤，不能同时定量活细胞、坏死细胞和凋亡细胞，以及非特异性检测（即“假阳性”）。为了克服目前的方法，在体外定量细胞凋亡的缺点，并利用96孔板格式，我们在这里提出了一种改进的溴化乙锭和吖啶橙子（EB/AO）染色测定，这可能是完全在96孔板进行。我们的方法结合了96孔格式和传统的EB/AO方法的优点，用于凋亡quantitation.Results：我们比较了我们的方法和传统的EB/AO方法，用于在正常生长和诱导凋亡条件下定量悬浮细胞（Jurkat）和贴壁细胞（A375）的凋亡。我们发现，我们的新EB/AO方法实现了定量结果相比，使用传统的EB/AO方法产生的悬浮液和贴壁cells.Conclusion：通过消除分离和洗涤步骤，我们的方法大大减少了所需的时间来执行测试，最大限度地减少对贴壁细胞的损伤，并降低了失去浮动细胞的可能性。总的来说，我们的方法是对目前可用技术的改进，特别是对于贴壁细胞。
Background: Analyzing apoptosis has been an integral component of many biological studies. However, currently available methods for quantifying apoptosis have various limitations including multiple, sometimes cell-damaging steps, the inability to quantify live, necrotic and apoptotic cells at the same time, and non-specific detection (i.e. "false positive"). To overcome the shortcomings of current methods that quantify apoptosis in vitro and to take advantage of the 96-well plate format, we present here a modified ethidium bromide and acridine orange (EB/AO) staining assay, which may be performed entirely in a 96-well plate. Our method combines the advantages of the 96-well format and the conventional EB/AO method for apoptotic quantification.Results: We compared our method and the conventional EB/AO method for quantifying apoptosis of suspension cells ( Jurkat) and adherent cells (A375) under normal growth and apoptosis-inducing conditions. We found that our new EB/AO method achieved quantification results comparable to those produced using the conventional EB/AO method for both suspension and adherent cells.Conclusion: By eliminating the detaching and washing steps, our method drastically reduces the time needed to perform the test, minimizes damage to adherent cells, and decreases the possibility of losing floating cells. Overall, our method is an improvement over the currently available techniques especially for adherent cells.