Quantitative analysis of cell proliferation by a dye dilution assay: Application to cell lines and cocultures

Cytometry A. 2017 Jul;91(7):704-712. doi: 10.1002/cyto.a.23105. Epub 2017 Apr 4.


Cell proliferation represents one of the most fundamental processes in biological systems, thus the quantitative analysis of cell proliferation is important in many biological applications such as drug screening, production of biologics, and assessment of cytotoxicity. Conventional proliferation assays mainly quantify cell number based on a calibration curve of a homogeneous cell population, and therefore are not applicable for the analysis of cocultured cells. Moreover, these assays measure cell proliferation indirectly, based on cellular metabolic activity or DNA content. To overcome these shortcomings, a dye dilution assay employing fluorescent cell tracking dyes that are retained within cells was applied and was diluted proportionally by subsequent cell divisions. Here, it was demonstrated that this assay could be implemented to quantitatively analyze the cell proliferation of different types of cell lines, and to concurrently analyze the proliferation of two types of cell lines in coculture by utilizing cell tracking dyes with different spectral characteristics. The mean division time estimated by the dye dilution assay is compared with the population doubling time obtained from conventional methods and values from literature. Additionally, dye transfer between cocultured cells was investigated and it was found that it is a characteristic of the cells rather than a characteristic of the dye. It was suggested that this method can be easily combined with other flow cytometric analyses of cellular properties, providing valuable information on cell status under diverse conditions. © 2017 International Society for Advancement of Cytometry.

Keywords: CFSE; cell division (doubling) time; cell lines; cell proliferation; coculture; dye dilution; dye transfer.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Assay* / methods
  • Cell Division / physiology
  • Cell Proliferation / physiology*
  • Cell Tracking / methods
  • Coculture Techniques* / methods
  • Dye Dilution Technique
  • Flow Cytometry* / methods
  • Fluoresceins / metabolism
  • Humans
  • Leukocytes, Mononuclear / cytology*


  • Fluoresceins