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Short answer
Cell cycle analysis by flow cytometry (often called cell cycle analysis FACS) measures how much DNA each cell carries. A DNA-binding dye stains cells stoichiometrically, so fluorescence tracks DNA content: cells in G0/G1 hold 2N DNA, cells in G2/M hold 4N, and cells in S phase fall between the two peaks. Software then converts the histogram into phase percentages.
Definition and scope
DNA-content cell cycle analysis is a population readout. It sorts cells into G0/G1, S and G2/M by how much DNA they contain, not by what they are doing. The plot is a histogram of cell count against linear fluorescence, and a 4N cell fluoresces about twice as brightly as a 2N cell (Thermo Fisher Scientific, cell cycle assays for flow cytometry). Because the readout is DNA mass, G2 and mitosis share one peak and G0 cannot be told from G1.
This page covers the stain-and-histogram approach. For the instrument itself see what a flow cytometer is; for gating and plot types see the flow cytometry principles guide; for one specific dye in depth see DAPI in flow cytometry.
How DNA-content cell cycle analysis works in practice
From stained cells to a histogram
Cells are stained with a DNA dye, run on the cytometer, and plotted as a one-parameter histogram. The cytometer is triggered on the dye signal. A core-facility protocol from UT Health San Antonio describes a primary gate on forward versus side scatter, then a second gate on pulse area versus pulse width to isolate single cells (UT Health San Antonio Flow Cytometry Core, cell cycle protocol, checked 2026-10-29).
Fixed versus live cells
Live-cell dyes need no fixation. Fixed-cell dyes are used after permeabilising fixation. The same core fixes cells in cold 70% ethanol and states that fixed samples may be kept for up to 2 weeks in 70% ethanol. Its reagent concentrations (RNase at 100 µg/ml, propidium iodide at 50 µg/ml) are that core’s own parameters; other cores use different ones, so follow your facility’s protocol. The core also flags propidium iodide as potentially mutagenic and calls for gloves.
Why propidium iodide is paired with RNase
Propidium iodide (PI) binds RNA as well as DNA. Thermo Fisher’s PI/RNase staining solution therefore includes RNase so that only DNA binding is observed.
How the percentages are produced
The phase percentages are model outputs, not counts read off the plot. The UT Health San Antonio core names ModFit (Verity Software House) and FlowJo, which fit a Gaussian curve to each of the G0-G1 and G2-M peaks and then calculate S phase.
Choosing a DNA stain
Thermo Fisher groups its cell cycle dyes by whether cells are live or fixed, and states a constraint for each. The table is organised by those stated constraints. It complements, rather than repeats, the DAPI/Hoechst/PI comparison on the DAPI guide.
| Stain | Live or fixed | Excitation / emission as stated | Constraint the manufacturer states |
|---|---|---|---|
| Hoechst 33342 | Live | UV or 405 nm | Needs a UV or violet laser |
| DRAQ5 | Live | 633 nm excitation, 697 nm emission | Described as cytotoxic and suboptimal for sorting; experiments under 2 h suggested |
| Vybrant DyeCycle (Violet, Green, Orange, Ruby) | Live | Colour set by variant | Non-toxic and suitable for sorting |
| DAPI | Live or fixed | Emission 452 nm when fixed | UV laser required for fixed cells |
| FxCycle Violet | Fixed | 405 nm excitation, 461 nm emission | Narrow emission, minimal compensation |
| Propidium iodide | Fixed | 488 or 532 nm excitation, 617 nm emission | Broad emission; binds RNA, so RNase is needed |
| 7-AAD | Fixed | Emission 647 nm | Broad emission; high compensation need |
| SYTOX AADvanced | Fixed | 488, 532 or 561 nm excitation, 647 nm emission | Takes up faster than 7-AAD |
| FxCycle Far Red | Fixed | 633 nm excitation, 658 nm emission | Suited to multiplexing |
Read the table by constraint: sorting live cells points to the non-toxic live dyes; a multicolour panel favours narrow-emission or far-red dyes that need little compensation (see fluorophore properties).
Why instrument QC and doublet gating decide the result
A cell-cycle histogram is only as trustworthy as the cytometer’s linearity and doublet discrimination. The UT Health San Antonio core describes two checks: chicken erythrocyte nuclei, used to set PMT voltage and gain and to check linearity and resolution, and calf thymocyte nuclei in the BD DNA QC kit (Cat. 349523), used to verify doublet-discrimination capability. Two G1 cells stuck together carry 4N DNA, so without pulse area versus width gating they read as G2/M. For daily setup practice, see instrument setup and QC beads.
A checklist for reading a cell-cycle histogram
- Trigger and display: the signal is the DNA dye, shown on a linear scale.
- Single cells only: pulse area versus width gate applied.
- Reference check: nuclei standards confirm linearity and doublet discrimination.
- RNase included when the dye is PI.
- Percentages come from a fitted model, so report the software used.
Common misunderstandings
“A high S-phase percentage means cells are proliferating”
DNA content does not show DNA synthesis directly. Thermo Fisher describes labelling with the thymidine analog EdU, detected by Click-iT chemistry and combined with a DNA dye such as FxCycle, as a more accurate S-phase measure because EdU selectively incorporates into the DNA of actively dividing cells. See the EdU assay guide.
“G2/M is the same as mitosis”
Both phases carry 4N DNA, so one peak holds G2 and M cells together.
“PI works without RNase”
PI binds RNA as well as DNA, which is why the vendor’s PI staining solution includes RNase.
“Any dye works on live cells”
Only the live-cell dyes in the table are described for unfixed cells.
FAQ
What is cell cycle analysis by flow cytometry?
It is the measurement of DNA content per cell with a DNA-binding dye, giving a histogram with a 2N (G0/G1) peak, a 4N (G2/M) peak and S-phase cells between them.
Does propidium iodide need RNase?
Yes. PI binds RNA as well as DNA, so RNase is included to isolate the DNA signal.
Can live cells be analysed?
Yes, with Hoechst 33342, DAPI, DRAQ5 or Vybrant DyeCycle dyes. DRAQ5 is described as cytotoxic; the Vybrant DyeCycle dyes as non-toxic and suitable for sorting.
How is S phase measured more accurately?
By EdU incorporation detected with Click-iT chemistry, combined with a DNA dye.
Sources
- Thermo Fisher Scientific, Cell cycle assays for flow cytometry, checked 2026-10-29.
- UT Health San Antonio Flow Cytometry Core, Cell Cycle Protocol (Propidium Iodide for DNA Content), checked 2026-10-29.
