BACKGROUND Whatever the target of an experiment in cell biology, cell counting and viability assessment are always computed. The Trypan Blue (TB) assay was proposed about a century ago and is still the most widely used method to perform cell viability analysis. Furthermore, the combined use of TB with a haemocytometer is also considered the standard approach to estimate cell population density. There are numerous research articles reporting the use of TB assays to compute cell number and viability of 2D and 3D cultures. However, the literature still lacks studies regarding the reliability of the TB assay in terms of assessment of its repeatability and reproducibility. METHODS We compared the TB assay's measurements obtained by two biologists who analysed 105 different samples in double-blind for a total of 210 counts performed. We measured: (a) the repeatability of the count performed by the same operator; (b) the reproducibility of counts performed by the two operators. RESULTS There were no significant differences in the results obtained with 2D and 3D cell cultures: we estimated an approximate variability of 5% when the TB assay was used to assess the viability of the culture, and a variability of around 20% when it was used to determine the cell population density. CONCLUSIONS The main aim of this study was to make researchers aware of potential measurement errors when TB is used with a haemocytometer for counting and viability measurements in 2D and 3D cultures. We believe that these results can help researchers to determine whether the expected reliability of the TB assay is compliant with their applications.

中文翻译：

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2D 和 3D 细胞培养物的细胞计数和活力评估：台盼蓝测定的预期可靠性。

背景技术无论细胞生物学实验的目标是什么，总是需要计算细胞计数和活力评估。台盼蓝 (TB) 测定法于大约一个世纪前提出，至今仍是最广泛使用的细胞活力分析方法。此外，TB 与血细胞计数器的结合使用也被认为是估计细胞群密度的标准方法。有许多研究文章报告了使用 TB 检测来计算 2D 和 3D 培养物的细胞数量和活力。然而，文献仍然缺乏关于结核病检测在重复性和再现性评估方面的可靠性的研究。方法 我们比较了两位生物学家获得的结核病检测测量结果，他们双盲分析了 105 个不同的样本，总共进行了 210 次计数。我们测量了： (a) 同一操作员执行计数的重复性；(b) 两名操作员执行的计数的再现性。结果 2D 和 3D 细胞培养物获得的结果没有显着差异：当使用 TB 测定来评估培养物的活力时，我们估计变异性约为 5%，而使用 TB 测定法时，变异性约为 20%以确定细胞群密度。结论 本研究的主要目的是让研究人员意识到当 TB 与血细胞计数器一起用于 2D 和 3D 培养物中的计数和活力测量时潜在的测量误差。我们相信这些结果可以帮助研究人员确定结核病检测的预期可靠性是否符合他们的应用。