A Quick Guide to Flow Cytometry Cell Cycle Analysis

Posted March 5, 2019

Cell Cycle

Flow cytometry cell cycle analysis typically involves using a DNA binding dye to determine each cell’s total DNA content. Sorting cells by cell cycle phase is then possible, as the total DNA content fluctuates as cells pass through G0/G1, S, and G2/M phases.

Cells in G2/M phase have replicated their DNA in preparation for mitotic division and will have twice as much DNA as cells in G0/G1 phase. Cells in S phase are in the process of DNA replication and will therefore have intermediate levels of DNA.

When choosing a DNA binding dye for your experiment, consider the following questions:

  1. Will you need a DNA specific dye? Some DNA binding dyes will also bind RNA, so RNase will be required to prevent false signals.
  2. Will your cells be fixed and permeabilized or do you plan to stain living cells? Some DNA binding dyes are membrane impermeant and require permeabilization.
  3. What colors dye do you need? There are many color options for DNA binding dyes! It may be prudent to design your multiparameter experiment by assigning dyes to uncommon markers first and then choosing your DNA dye color based on your cytometer’s remaining channels.

Use FluoroFinder’s antibody search tool to find conjugated antibodies from any supplier and compare fluorophores side-by-side on the spectraviewer.

Use the following table of DNA binding dyes to find the best option for your experiment.


Name Excitation Emission Cell Permeant? DNA Specific? Links
Vybrant DyeCycle Violet 369nm 437nm Yes Yes View Products
Hoechst (34580) 392nm 440nm Yes Yes View Products
CytoPhase™ Violet 440nm 440nm Yes Yes
DAPI (4′,6-diamidino-2-phenylindole) 358nm 461nm No Yes View Products
Hoechst (33258) 352nm 461nm Yes Yes View Products
Hoechst (33342) 361nm 497nm Yes Yes View Products
YOYO-1 491nm 509nm No No View Products
TOTO-1 514nm 533nm No No View Products
Vybrant DyeCycle Green 506nm 534nm Yes Yes View Products
Vybrant DyeCycle Orange 519nm 563nm Yes Yes View Products
Propidium Iodide (PI) 535nm 617nm No No View Products
YOYO-3 612nm 631nm No No View Products
7-AAD (7-aminoactinomycin D) 546nm 647nm No Yes View Products
TOTO-3 642nm 660nm No No View Products
Vybrant DyeCycle Ruby 638nm 686nm Yes Yes View Products
DRAQ5 633nm 695nm Yes Yes View Products
DRAQ7 633nm 695nm No Yes View Products


For experiments where researchers need to further distinguish between G0 and G1 or G2 and M phases, cell cycle specific markers can be used in combination with the DNA stain.

G0 cells are quiescent or arrested and lack the cell cycle machinery necessary to begin the cell cycle. Therefore, negative staining for cell cycle machinery markers, such as cyclin D1 [PMID: 12648671] or cyclin E [PMID: 1833068], may identify cells in G0 phase. Additionally, quiescent G0 cells transcribe RNA at much lower levels than G1 cells, so RNA staining may also be used to distinguish between these two phases.

Cells in both G2 and M phases have twice as much DNA as G0/G1 cells. Fortunately, there are several markers that can help distinguish between them. Histone 3 becomes phosphorylated in mitotic cells between prophase and telophase, so phospho-H3 specific markers can identify M phase cells. Alternatively, Cyclin A is expressed in late S and G2 phases but is absent during mitosis.

Once again, the cell cycle machinery marker Cyclin E may be used to separate early and late S phase cells, as its expression decreases from early to late S phase.

In summary, DNA binding dyes and cell cycle specific markers can be used to distinguish cells in each phase, giving researchers a powerful tool to understand how their cells are growing and replicating.

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