Cell Cycle Analysis by Flow Cytometry

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 the DNA content distribution across the steps of the cell cycle (Figure 1A). 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 (Figure 1B).

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 (Figure 2). This method provides a more accurate quantitation of the S phase because the thymidine analogue selectively incorporates into DNA of actively dividing cells.

We offer classic DNA cell cycle stains such as Hoechst 33342 and DRAQ5 for cell cycle analysis, but most of these have limitations that have to be considered when using them in an experiment (see Selection Guide for live-cell cycle stains) which is why the Invitrogen Vybrant DyeCycle stains for live-cell cycle analysis were developed.

Vybrant DyeCycle stains are:

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

Figure 3. Brightfield images of cells grown after sorting. Live Jurkat cells were stained with either 5 µM UV-excitable Hoechst 33342, 5 µM Vybrant DyeCycle Ruby stain, or 5 µM DRAQ5 DNA dye. The cells were then sorted and cultured for 8 days to examine their ability to grow after staining and sorting. Visual inspection of the cultures shows the characteristic grape-like clusters of growing Jurkat cells, in the cells stained with Hoechst 33342 and Vybrant DyeCycle Ruby Stains, comparable to the control cells. The cells stained with DRAQ5 dye did not show appreciable growth, indicating a much higher level of cytotoxicity in that treatment.

*These reagents are also available in the ready-to-use, Ready Flow format that are designed to allow you to stain your cells without the need for calculations, dilutions or pipetting.

Combining cell cycle analysis with additional live cell applications (i.e., analysis of GFP cells, immunophenotyping, cell sorting, CFSE cell tracing, and apoptotic sub-G1 population analysis) is simple with Vybrant DyeCycle Stains. These stains are cell-permeant nucleic acid stains that can penetrate the nucleus without cell fixation.

If Vybrant DyeCycle stains are used in combination with other stains for multicolor applications, apply the other stain(s) to the sample first, following all manufacturers’ instructions, including wash steps. The DyeCycle stain should be the last stain applied to the sample, and do not wash or fix samples prior to flow cytometric analysis.

Vybrant DyeCycle Violet Stain has been shown to identify side populations (CD34^– cells) in stem cells and thus, allow for a phenotype by which to isolate this type of cell population (Figure 4). The basis of the side population technique is that human and rodent stem cells efflux Vybrant DyeCycle Violet stain (and also Hoechst 33342 stain). The efflux can be blocked with verapamil, fumitremorgin C, or other such blocking agents, to prevent dye efflux for accurate DNA content analysis in these stem cells.

In Current Protocols in Cytometry in 2013 [5-7], the Petriz lab demonstrated that isolated cells using DyeCycle Violet were shown to have the same phenotypic characteristics of side population cells isolated with Hoechst 33342.

Figure 4. Identification of stem cells based on differential efflux of Vybrant DyeCycle Violet dye in human adenocarcinoma cells. (A) Staining of A549 cells with 5 μM Vybrant DyeCycle Violet results in a poor DNA content histogram because the dye is actively pumped out of cells by the ABCG2 membrane pump. (B) Treatment of A549 cells with the ABCG2 inhibitor fumitremorgin C (10 μM) results in a typical DNA content histogram indicating retention of stain in the cells. (C, D) Vybrant DyeCycle Violet has a broad fluorescence emission which can be detected. Dual-parameter plots of VL1 vs. VL3 provide better discrimination between cells of the side population phenotype, those actively effluxing Vybrant DyeCycle Violet Stain and displaying the side population tail (C), and cells treated with the ABCG2 membrane pump inhibitor fumitremorgin C (D).

We offer classic DNA cell cycle stains such as DAPI, PI, and 7-AAD for fixed cell cycle analysis, but these reagents do not cover the full spectrum of laser excitation available. The FxCycle reagents 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. See the Selection Guide for fixed-cell cycle stains.

FxCycle stains provide:

• Flexibility—Options for 405 and 633 nm laser excitation to increase multiplexability in cell cycle studies
• Tight CVs—More accurate analysis due to a narrow emission spectra requiring very minimal compensation

*The FxCycle Violet dye is also available in the ready-to-use, Ready Flow format that is designed to allow you to stain your cells without the need for calculations, dilutions, or pipetting.