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.
Why flow cytometry is ideal for cell cycle analysis
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.
Figure 1. Flow cytometry DNA content distribution in a cell cycle analysis assay. (A) Histogram of live Jurkat cells stained with Vybrant DyeCycle Violet stain showing DNA content distribution. G0/G1 and G2/M phase histogram peaks are separated by the S-phase distribution. Violet 405 nm excitation was used with a 440 nm bandpass filter. (B) Modeling of the expected DNA distribution based on linear fluorescence and stoichiometric staining. The fluorescence of the 4N cells at G2M is twice the 2N cells at G0/G1.
Figure 2. Cell cycle analysis with FxCycle Violet Ready Flow Reagent 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%.
Live-cell cycle analysis stains—Vybrant DyeCycle stains
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.
Live-cell cycle stains selection guide
|Classic Dyes||Optimized Vybrant DyeCycle Stains|
|Hoechst 33342*||DRAQ5||DyeCycle Violet*||DyeCycle Green||DyeCycle Orange||DyeCycle Ruby|
|Laser excitation (nm)||UV, 405||633||UV, 405||488||488, 532||633|
|Experiment length||Short or long-term||Short-term are best (<2 hours)||Short or long-term|
|Considerations||UV light may have a detrimental effect on living cells
Can be used for stem cell side population studies [1-3]
|Cytotoxicity is an issue in live-cell analysis||Additional color options for live-cell analysis
Ideal for cell sorting applications
DyeCycle violet has tighter CVs compared to Hoechst when excited off the 405 nm laser
DyeCycle Violet can be used for stem cell side population studies [5-7]
|Assays or amount||10 mL||200 uL||200 assays||200 assays||200 assays||100 assays|
*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.
Easily combine DyeCycle stains with other live-cell applications
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.
Identify stem cell side populations using DyeCycle Violet
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, fumitermorgin 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).
Fixed-cell cycle analysis stains—FxCycle reagents
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 multiplexabilty in cell cycle studies
- Tight CVs—More accurate analysis due to a narrow emission spectra requiring very minimal compensation
Fixed-cell cycle reagents selection guide
|DAPI||FxCycle Violet*||7-AAD||SYTOX AADvanced||PI||FxCycle PI/RNase||FxCycle Far Red|
|Laser excitation (nm)||UV||405||488, 532||488, 532, 561||488, 532||488, 532||633|
|Considerations||UV laser required||Narrow emission spectra
|Broad emission spectra
High compensation required
|Similar spectra to 7-AAD but faster uptake
Provides better separation of live/dead cells
|Broad emission spectra
Binds DNA and RNA
|Contains RNAse to keep PI from binding RNA
Observe only DNA binding
|633 nm laser excitation, ideal for multiplex analyses|
|Assays or amount||1 mL||500||2 mL||100||500||10 mL||200||500|
*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.
FxCycle stains flow cytometry data
Figure 5. Multiparametric cell cycle and immunophenotypic analysis. TF-1 erythroblast cells were alcohol-fixed overnight, washed, and then suspended in 0.1% Triton X-100/PBS/1% BSA before staining with anti–histone H3[pS10] purified antibody complexed with Zenon Alexa Fluor 488 Rabbit IgG labeling reagent and FxCycle Violet stain. The pH3 signal (red) identifies cells that are in mitosis.
Figure 6. A mixed population of Jurkat cells were stained with SYTOX AADvanced Dead Cell Stain Kit and analyzed by flow cytometry. A mixture of heat-killed and untreated Jurkat cells were stained with 1 uM SYTOX AADvanced Dead Cell Stain Kit for 5 minutes. Cells were analyzed on a flow cytometer equipped with a 488 nm laser and a 695/40 nm bandpass filter. Live cells are easily distinguished from the dead cell population.
Figure 7. Histogram of Jurkat cells stained with FxCycle PI/RNase stain showing DNA content distribution. Jurkat cells were fixed in 70% ethanol, washed, and then resuspended in FxCycle PI/RNase stain for 30 minutes at room temperature. G0/G1 and G2/M phase histogram peaks are separated by the S phase distribution. Analysis was performed using 532-nm excitation with a 585/42-nm bandpass filter.
Figure 8. Histogram of TF-1 erythroblast cells stained with FxCycle Far Red stain showing DNA content distribution. TF-1 cells were fixed overnight with alcohol, washed, and then resuspended in 0.1% Triton X-100/PBS/1% BSA before staining with FxCycle Far Red stain plus RNase A for 30 minutes at room temperature. G0/G1 and G2/M phase histogram peaks are separated by the S-phase distribution. Analysis was performed using 633 nm excitation with a 660/20 bandpass filter.
- Petriz J. (2013) Flow cytometry of the side population (SP) Curr Protoc Cytom. 64: 9.23.1-9.23.20.
- Goodell MA. (2005) Stem cell identification and sorting using the Hoechst 33342 side population (SP). Curr Protoc Cytom. 9.18.
- Telford WG, Frolova EG. (2004) Discrimination of the Hoechst side population in mouse bone marrow with violet and near-ultraviolet laser diodes. Cytometry A. 57(1):45-52.
- Pastrana E, Cheng L-C, Doetsch F (2009) Simultaneous prospective purification of adult subventricular zone neural stem cells and their progeny. PNAS 106 (15): 6387-92.
- Telford WG, (2010) Stem Cell Side Population Analysis and Sorting Using DyeCycle Violet. Curr Protoc Cytom. 51: 9.30.1-9.30.9.
- Gangavarpu KJ, Huss WJ (2011) Isolation and Applications of Prostate Side Population Cells Based on Dye Cycle Violet Efflux. Curr Protoc Tox 47: 22.2.1-22.2.14.
- Telford WG, (2013) Stem Cell Identification by DyeCycle Violet Side Population Analysis. Basic Cell Culture Protocols: 163-179.
- Petriz J. (2017) Cancer Stem Cells and Multi-drug Resistance by Flow Cytometry. In: Robinson J., Cossarizza A. (eds) Single Cell Analysis. Series in BioEngineering. Springer, Singapore.
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