Filby Andrew, Begum Julfa, Jalal Marwa, Day William
Flow Cytometry Core Facility, Newcastle Biomedicine, Newcastle University, Newcastle-upon-Tyne NE1 7RU, UK; FACS Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, Holborn, WC2A 3LY London, UK.
FACS Laboratory, London Research Institute, Cancer Research UK, 44 Lincoln's Inn Fields, Holborn, WC2A 3LY London, UK.
Methods. 2015 Jul 1;82:29-37. doi: 10.1016/j.ymeth.2015.02.016. Epub 2015 Mar 20.
Successful completion of the cell cycle usually results in two identical daughter progeny. This process of generational doubling is termed proliferation and when it occurs in a regulated fashion the benefits range from driving embryonic development to mounting a successful immune response. However when it occurs in a dis-regulated fashion, it is one of the hallmarks of cancer and autoimmunity. These very reasons make proliferation a highly informative parameter in many different biological systems. Conventional flow cytometry (CFC) is a high-throughput, fluorescence-based method for measuring the phenotype and function of cells. The application of CFC to measuring proliferation requires a fluorescent dye able to mark live cells so that when they divide, the daughter progeny receives approximately half the fluorescence of the parent. In measurement space, this translates into peaks of fluorescence decreasing by approximately half, each corresponding to a round of division. It is essential that these peaks can be resolved from one another otherwise it is nearly impossible to obtain accurate quantitative proliferation data. Peak resolution is affected by many things, including instrument performance, the choice of fluorescent dye and the inherent properties of the cells under investigation. There are now many fluorescent dyes available for tracking proliferation by dye dilution differing in their chemistry and spectral properties. Here we provide a method for assessing the performance of various candidate dyes with particular emphasis on situations where the cell type is non-quiescent. We have shown previously that even under optimised instrument and labelling conditions, the heterogeneity of non-quiescent cells makes it impossible to obtain an input width below the threshold for peak resolution without reducing the fluorescence distribution using a cell sorter. Moreover, our method also measures how the dye performs post-labelling in terms of loss/transfer to other cells and how the dye is inherited across the cytokinetic plane. All of these factors will affect peak resolution both in non-quiescent and primary cell types.
细胞周期的成功完成通常会产生两个相同的子代细胞。这种世代翻倍的过程被称为增殖，当它以一种受调控的方式发生时，其益处涵盖从驱动胚胎发育到引发成功的免疫反应等多个方面。然而，当增殖以不受调控的方式发生时，它是癌症和自身免疫的标志之一。正是这些原因使得增殖在许多不同的生物系统中成为一个极具信息量的参数。传统流式细胞术（CFC）是一种基于荧光的高通量方法，用于测量细胞的表型和功能。将CFC应用于测量增殖需要一种能够标记活细胞的荧光染料，以便当细胞分裂时，子代细胞接收的荧光约为亲代细胞的一半。在测量空间中，这表现为荧光峰值大约减半，每个峰值对应一轮细胞分裂。这些峰值必须能够相互区分，否则几乎不可能获得准确的定量增殖数据。峰值分辨率受许多因素影响，包括仪器性能、荧光染料的选择以及所研究细胞的固有特性。现在有许多荧光染料可用于通过染料稀释追踪增殖，它们在化学性质和光谱特性上有所不同。在这里，我们提供一种评估各种候选染料性能的方法，特别强调细胞类型为非静止细胞的情况。我们之前已经表明，即使在优化的仪器和标记条件下，非静止细胞的异质性使得在不使用细胞分选仪降低荧光分布的情况下，无法获得低于峰值分辨率阈值的输入宽度。此外，我们的方法还测量了染料在标记后的表现，包括其向其他细胞的损失/转移情况以及染料在细胞分裂平面上的遗传方式。所有这些因素都会影响非静止细胞类型和原代细胞类型中的峰值分辨率。
