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. Author manuscript; available in PMC: 2018 Feb 8.
Published in final edited form as: Cytometry A. 2016 Feb 3;89(3):233–235. doi: 10.1002/cyto.a.22815

Table 1. Proliferation Monitoring by Flow Cytometry: Content Overview.

Module Target Audience Topics
Lesson 1 Basics of the Cell Cycle Individuals with a basic understanding of flow cytometry who are unfamiliar with, or wish to review, the biology of the cell cycle.

  • Phases of the cell cycle and molecular events associated with each

  • Cell cycle checkpoints and regulators (cyclins and cyclin dependent kinases)

  • Relationship between the cell cycle phase distribution of a sample and DNA content histogram

  • Use of DNA content histograms in combination with metabolic labeling or cellular synchronization to determine kinetics of progression through the different cell cycle phases
Lesson 2 Monitoring Cell Cycle Progression Individuals with a basic understanding of flow cytometry who are unfamiliar with, or wish to review, how flow cytometry is used to monitor the distribution or progression of cells through the cell cycle.
  • Principles and methods for the use of DNA binding dyes alone, or in combination with thymidine analogs, proliferation related antigens, and/or phase specific expression vectors, to assess:
    1. How cells within a population of interest are distributed among the different cell cycle phases; and
    2. The kinetics with which cells of interest are progressing through the cell cycle.
  • Strategies for analysis and interpretation of single and dual parameter DNA histograms to determine:
    1. The fraction of cells actively synthesizing DNA; and
    2. Whether a cell population is non-cycling, actively progressing through the cell cycle or blocked in a specific phase of the cell cycle.

  • Practical considerations, common pitfalls, critical controls and troubleshooting tips

  • Applications of DNA content analysis andcell cycle progression kinetics to:tumor diagnosis and prognosis; anti-tumormechanisms of therapeutic agents; and regulation of quiescence or senescence
Lesson 3 Monitoring Cell Division by Dye Dilution Individuals with some hands-on experience with flow cytometry who are unfamiliar with, or wish to review, how flow cytometry is used to estimate the number of cell generations and daughter cells in each generation without manual counting

  • Principles and methods for the use of protein-reactive and/or membrane-intercalating dyes to assess extent of cell division in cell subsets of interest based on dye dilution

  • Similarities, differences and considerations for selecting among commercially available “proliferation tracking dyes”

  • Strategies for analysis and interpretation of dye dilution profiles to estimate:
    1. Number of generations present at the end of a test and number of daughter cells in each
    2. Total population expansion during the test period and frequency of responder cells in the starting population

  • Practical considerations, common pitfalls, critical controls and troubleshooting tips

  • Applications of dye dilution proliferation analysis to identification of: immunosuppressive effector cell potency; differential proliferative responses among immune cell subsets; whether/how early activation markers (antigen binding, CD69, cytokine expression) correlate with subsequent cell division; effective vaccination protocols for increasing precursor cell frequency; and quiescent or slow growing cell subpopulations with stem-like properties