Appraising the suitability of succinimidyl and lipophilic fluorescent dyes to track proliferation in non-quiescent cells by dye dilution

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.