Adaptation of stem cells to 96-well plate assays: use of human embryonic and mouse neural stem cells in the MTT assay.
Adaptation of stem cells to 96-well plate assays: use of human embryonic and mouse neural stem cells in the MTT assay.
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DOI：
10.1002/9780470151808.sc01c13s23
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发表时间：
2012-11-01
影响因子：
--
通讯作者：
Talbot, Prue
中科院分区：
文献类型：
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作者：
Behar, Rachel Z;Bahl, Vasundhra;Talbot, Prue
人胚胎干细胞（hESC）很难适应96孔板试验，如MTT试验，因为它们在作为菌落电镀时存活得最好，而菌落不容易计数和准确电镀。为了解决这个问题，开发了两种方法。首先，使用ROCK抑制剂（ROCKi），可以对单个hESC进行精确计数和电镀。在第二个实验中，小的菌落没有被镀上ROCKi，但有精确计数和镀上的适应性。MTT试验也适用于小鼠神经干细胞。这些方法使MTT测定能够快速准确地进行，在重复实验之间具有很高的再现性。在96孔板中筛选挥发性化学物质时，可能会发生蒸气效应，必须仔细确定剂量范围。采用NIH检测指导工具对方法进行验证。这些方法可以很容易地推广到其他96孔板试验中。
Human embryonic stem cells (hESC) are difficult to adapt to 96-well plate assays, such as the MTT assay, because they survive best when plated as colonies, which are not easily counted and plated accurately. Two methods were developed to address this problem. In the first, ROCK inhibitor (ROCKi) was used, which allows accurate counting and plating of single hESC. In the second, small colonies were plated without ROCKi but with adaptations for accurate counting and plating. The MTT assay was also adapted for use with mouse neural stem cells. These methods allow the MTT assay to be conducted rapidly and accurately with high reproducibility between replicate experiments. When screening volatile chemicals in a 96-well plate, vapor effects may occur and dose ranges must be carefully defined. The methods were validated using the NIH assay guidance tool. These methodss could readily be translated to other 96-well plate assay.