包括增殖染料的高维研究在面板设计中面临两个固有的挑战。首先,基于染料稀释监测的细胞分裂次数越多,标记的亲本细胞的起始强度必须越大,以便将高度分裂的子细胞与背景自发荧光区分开。第二,他们的起始强度越大,越难以避免增殖染料信号溢出到相邻的光谱通道,对其他荧光染料的使用和分辨感兴趣的昏暗信号的能力产生限制。在本系列的第三和第四版中,我们描述了用于一般细胞示踪的蛋白质反应染料和膜嵌入染料之间的异同,提供了每种染料类型的优化标记的详细方案,以及在验证任一种染料类型用作增殖染料时由供应商和/或用户测试的总结特征。在第五版中,我们回顾:(a)染料稀释增殖测定的基本假设和关键对照;(b)评估标记对细胞生长速率的影响和测试染料稀释报告细胞分裂的保真度的方法;和。(c)确定有多少子代可以准确地包括在增殖建模中的因素。我们还提供了关于光谱表征的扩展部分,使用收集的三种蛋白质活性染料的数据(CellTrace™Violet,CellTrace™CFSE,和CellTrace™FarRed)和三种膜嵌入染料(PKH67,PKH26和CellVue®Claret)在三个不同的细胞仪上,以说明多色面板设计期间所需的典型决策和权衡。最后,我们包括评估调节性T细胞效力的方法和对照,包含本文所述的“知道你的染料”和“知道你的细胞仪”原理的功能测定。
High dimensional studies that include proliferation dyes face two inherent challenges in panel design. First, the more rounds of cell division to be monitored based on dye dilution, the greater the starting intensity of the labeled parent cells must be in order to distinguish highly divided daughter cells from background autofluorescence. Second, the greater their starting intensity, the more difficult it becomes to avoid spillover of proliferation dye signal into adjacent spectral channels, with resulting limitations on the use of other fluorochromes and ability to resolve dim signals of interest. In the third and fourth editions of this series, we described the similarities and differences between protein-reactive and membrane-intercalating dyes used for general cell tracking, provided detailed protocols for optimized labeling with each dye type, and summarized characteristics to be tested by the supplier and/or user when validating either dye type for use as a proliferation dye. In this fifth edition, we review: (a) Fundamental assumptions and critical controls for dye dilution proliferation assays; (b) Methods to evaluate the effect of labeling on cell growth rate and test the fidelity with which dye dilution reports cell division; and. (c) Factors that determine how many daughter generations can be accurately included in proliferation modeling. We also provide an expanded section on spectral characterization, using data collected for three protein-reactive dyes (CellTrace™ Violet, CellTrace™ CFSE, and CellTrace™ Far Red) and three membrane-intercalating dyes (PKH67, PKH26, and CellVue® Claret) on three different cytometers to illustrate typical decisions and trade-offs required during multicolor panel design. Lastly, we include methods and controls for assessing regulatory T cell potency, a functional assay that incorporates the \"know your dye\" and \"know your cytometer\" principles described herein.