Viability Assays for Cells in Culture
Viability Assays for Cells in Culture
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DOI：
10.3791/50645
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发表时间：
2014-01-01
影响因子：
1.2
通讯作者：
Leak, Rehana K.
中科院分区：
文献类型：
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作者：
Posimo, Jessica M.;Unnithan, Ajay S.;Leak, Rehana K.
显微镜上的人工细胞计数是评估细胞活性的一种敏感手段，但很耗时，因此成本高昂。就设备而言，计算机化的细胞活力分析是昂贵的，但可以比人工细胞计数更快、更客观。本报告描述了三种这样的生存能力分析的使用。这些分析中有两种是红外的，一种是发光的。这两种红外分析都依赖于16位的奥德赛成像仪。一种红外分析使用DRAQ5染色进行细胞核染色，结合蓝宝石染色进行胞浆染色，并在700 nm通道中可见。另一种红外分析是In-Cell Western，它使用针对细胞骨架蛋白(α-微管蛋白或微管相关蛋白2)的抗体，并在800 nm的通道中标记它们。第三种活力测定法是一种常用的ATP发光测定法，但为了节省成本，我们使用了推荐体积的四分之一。这些测量都是线性的，并与电镀细胞的数量相关，但灵敏度不同。所有这三种分析方法都避开了耗时的显微镜检查，并对整个油井进行采样，从而减少了采样误差。最后，所有的分析可以在实验结束后的一天内轻松完成，从而允许在短时间内进行更多的实验。然而，它们都依赖于细胞数量与治疗后信号强度成正比的假设，这一假设有时不能满足，特别是对细胞内的ATP来说。此外，如果细胞在治疗后增大或减小，这可能会影响信号强度，而不影响细胞数量。我们的结论是，所有的活性分析，包括人工计数，都有一些需要注意的地方，但计算机化的活性分析非常值得最初的投资。综合使用这三种分析方法，可以全面了解细胞的结构和功能。
Manual cell counts on a microscope are a sensitive means of assessing cellular viability but are time-consuming and therefore expensive. Computerized viability assays are expensive in terms of equipment but can be faster and more objective than manual cell counts. The present report describes the use of three such viability assays. Two of these assays are infrared and one is luminescent. Both infrared assays rely on a 16 bit Odyssey Imager. One infrared assay uses the DRAQ5 stain for nuclei combined with the Sapphire stain for cytosol and is visualized in the 700 nm channel. The other infrared assay, an In-Cell Western, uses antibodies against cytoskeletal proteins (alpha-tubulin or microtubule associated protein 2) and labels them in the 800 nm channel. The third viability assay is a commonly used luminescent assay for ATP, but we use a quarter of the recommended volume to save on cost. These measurements are all linear and correlate with the number of cells plated, but vary in sensitivity. All three assays circumvent time-consuming microscopy and sample the entire well, thereby reducing sampling error. Finally, all of the assays can easily be completed within one day of the end of the experiment, allowing greater numbers of experiments to be performed within short timeframes. However, they all rely on the assumption that cell numbers remain in proportion to signal strength after treatments, an assumption that is sometimes not met, especially for cellular ATP. Furthermore, if cells increase or decrease in size after treatment, this might affect signal strength without affecting cell number. We conclude that all viability assays, including manual counts, suffer from a number of caveats, but that computerized viability assays are well worth the initial investment. Using all three assays together yields a comprehensive view of cellular structure and function.