Cell Cycle Assays for Flow Cytometry

Flow cytometry can be used for cell cycle analysis to estimate the percentages of a cell population in the different phases of the cell cycle, or it can be used with other reagents to analyze just the S phase.

On this page:

• Cell cycle live-cell cycle stains selection guide
• Cell cycle fixed-cell cycle stains selection guide
• Flow cytometry cell cycle analysis
• Vybrant DyeCycle stains for live-cell cycle analysis
• FxCycle stains for fixed-cell cycle analysis

We offer classic DNA cell cycle stains such as Hoechst 33342 and DRAQ5 for live-cell cycle analysis. In addition, Vybrant DyeCycle stains for live-cell cycle analysis are also available. Vybrant DyeCycle stains alleviate some of the limitations that traditional stains impose. Use the table below to select a live-cell stain and review its characteristics.

We offer classic DNA cell cycle stains such as DAPI, PI, and 7-AAD for fixed-cell cycle analysis. In addition, FxCycle stains for fixed-cell cycle analysis are also available. FxCycle Stains allow for multiplexing and more accurate analysis. Use the table below to select a fixed-cell cycle stain and review its characteristics.

Explore: Additional Cell Cycle Analysis Assays

When stained with a cell cycle reagent, DNA in the cells bind the dye stoichiometrically (in proportion to the amount of DNA present in each cell). The flow cytometric analysis of cell count versus linear fluorescence is used to create a histogram of DNA content distribution across the steps of the cell cycle. There are standard modeling algorithms that can then be employed to determine the breakdown of cells in the G0/G1 phase versus S phase, G2, or polyploidy state of the cell population.

Alternatively, the S phase can be detected in fixed cell applications using a dual-labeling experiment in which the DNA of proliferating cells are labeled with EdU (a thymidine analog), detected with a Click-iT EdU Assay and subsequently stained with a DNA dye such as the FxCycle stain. This method provides a more accurate quantitation of the S phase because the thymidine analogue selectively incorporates into DNA of actively dividing cells.

Figure 2. Cell cycle analysis with FxCycle Violet Stain and Invitrogen Click-iT EdU Alexa Fluor 647 Flow Cytometry Assay Kit. Jurkat cells, a human T cell leukemia cell line, were pulsed with 10 µM EdU for 2 hours prior to detection with Alexa Fluor 647 azide. Cells were subsequently stained by adding 2 drops of FxCycle Violet Ready Flow Reagent and incubated for 30 minutes, at 25°C. Data was acquired on an Invitrogen Attune NxT Flow Cytometer using a 405 laser and 440/50 nm emission filter. Analysis of the population indicates the following distribution: apoptotic sub-G1 cells were 3.4%; G0/G1, 49.1%; S 33.0%; and G2M 14.0%.

Vybrant DyeCycle stains are cell permeable DNA dyes for live-cell cycle analysis by flow cytometry without cell fixation. They help to eliminate the limitations of traditional stains.

Vybrant DyeCycle stains advantages include:

• Precise cell cycle analysis in living cells
• Low cytotoxicity for combining with additional live cell experiments
• Easily sort cells based on phase of the cell cycle
• Stains available for all common laser lines (UV, 405, 488, 532, and 633 nm)

In addition, Vybrant DyeCycle stains allow for the ability to combine cell cycle analysis with additional live cell applications (i.e., GFP cell analysis, immunophenotyping, cell sorting, CFSE cell tracing, and apoptotic sub-G1 population analysis). When using Vybrant DyeCycle stains in combination with other stains for multicolor applications, apply the other stains to the sample first (following all manufacturers’ instructions, including wash steps) and apply the Vybrant DyeCycle stain last. There is no need to wash or fix the sample prior to flow cytometric analysis.

Protocol: Vybrant DyeCycle stains

We offer classic DNA cell cycle stains for fixed-cell cycle analysis; however, they don’t cover the full spectrum of laser excitation. FxCycle stains offer options for the 405 nm (violet) and 633 nm (red) laser thereby increasing the ability to multiplex by freeing up the 488 nm and 633 nm lasers for other cellular analyses such as immunophenotyping, apoptosis analysis, and dead cell discrimination.

FxCycle stains advantages include:

• Stain options available for 405 and 633 nm laser excitation to increase multiplex ability in cell cycle studies
• More accurate analysis due to a narrow emission spectra requiring minimal compensation