Table 2.

A comparison of common and emerging techniques to study cell cycle dynamics.

Method	Use/Readout	Advantages	Limitations
DNA content dye	Snapshot of cell cycle phase distribution	• Rapid protocol • 1-time measurement • Live-cell dyes available • Enables FACS sorting	• Population level • Not a quantitative measurement of cell cycle phase duration
pH3 or single thymidine pulse	Frequency of cells in M-phase (pH3) or S-phase (thymidine analog)	• Robust, specific labeling • 1-time measurement • Acceptable for qualitatively comparing proliferation rates between populationsa	• Population level • Not a quantitative measurement of cell cycle phase duration • Requires fixation
Cell counting time course	Measures population growth over time	• Straightforward protocol • Reliable quantification of doubling time (when combined with an assay to measure cell death rate)	• Impractical or currently not possible in vivob • Doubling time represents population average • Multiple measurements
Live cell cycle phase reporter	Monitors cellular status with respect to a particular cell cycle phase	• Live cell assay • Continuous monitor • Single-cell resolution (when combined with time-lapse microscopy) • Most reporters enable FACS	• Single snapshot yields limited inferences about cell cycle dynamics (similar to DNA dyes or fixed-cell cycle markers)
Time-lapse microscopy	Direct visualization of live cells over time	• Definitive, ground-truth measurement of cell cycle rates • Single-cell resolution • Spatial information retained • Allows assessment of other cellular features, for example, morphology and migration	• Large data files • Time-intensive postacquisition processing/analysis • Single-cell tracking could be difficult for some cell types • Challenging or currently limited in vivob • Information gained is retrospective • Conditions require careful optimization (e.g., temperature, gas, phototoxicity, evaporation)
2-Thymidine assay	Quantification of S-phase duration and cell cycle period	• Single snapshot • Allows cell cycle length quantification in otherwise inaccessible tissues	• Difficult protocol requiring many starting cells • Population average • Validity depends on population homogeneity • Requires fixation
Fluorescent dye retention assay	Identification of cells which have undergone one or more divisions based on dilution of a fluorescent dye label	• Enables FACS-based prospective isolation of cells with different cycling rates • Does not require cloning or transgenic animals	• Limited dynamic range • Limited sensitivity for cells that are too homogeneous • Moderate cytotoxicity • Multiple measurements requiring 2 rounds of single-cell dissociation
Genetic label retention assay	Identification of nondividing cells based on retention of a stable fluorescent protein after expression is turned off	• Positively identifies nondividing label retaining cells, including rare populations in vivo that can be otherwise difficult to mark • Used in live animals	• Dividing cells don’t form well-resolved peaks, limiting sensitivity and dynamic range • Not a continuous monitor of cell cycle dynamics
H2B-FT reporter	Ratiometric reporter of cell cycle speed	• Single snapshot • Single-cell resolution • Enables FACS-based prospective isolation of cells with different cycling rates • Useful in live cells and animals • Continuously responsive to changes in cell division rate • Allows quantification of cell cycle rate for fast-cycling cells (<30h per cellcycle)	• Resolution among slow-cycling cells limited (only resolvable up to approximately 100h per cell cycle) • Occupies two fluorescent channels • Intensity must be captured in fresh tissue prior to fixation (blue form of FT is converted to red upon exposure to fixative) • Blue FT is photo-convertible, so z-stack imaging not enabled

^aWith caveats described in main text.

^bDepending on model organism and whether the tissue is accessible for intravital microscopy.