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 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)
4 panels. First 3 panels all show healthy cells. DRAQ5 panel shows many fewer cells.

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
Emission (nm) 461 697 437 534 563 686
Toxicity Non-toxic Cytotoxic Non-toxic Non-toxic Non-toxic Non-toxic
Cell sorting Yes Suboptimal Yes Yes Yes Yes
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
Cat. No. H3570 65-0880-92 V35003 V35004 V35005 V10309

*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
Emission (nm) 452 461 647, 647 617 617 658
Considerations UV laser required Narrow emission spectra

Minimal compensation
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
Cat No. 62248 F10347 00-6993-50 S10349 S10274 P3566 F10797 F10348

*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

FxCycle Violet Stain

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.

SYTOX AADvanced Dead Cell Stain
FxCycle PI/RNase Staining Solution

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.

FxCycle Far Red stain
References
  1. 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.
  2. Telford WG, (2010) Stem Cell Side Population Analysis and Sorting Using DyeCycle Violet. Curr Protoc Cytom. 51: 9.30.1-9.30.9.
  3. 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.
  4. Telford WG, (2013) Stem Cell Identification by DyeCycle Violet Side Population Analysis. Basic Cell Culture Protocols: 163-179.
  5. 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.
  6. Petriz J, Bradford JA, Ward MD (2018) No lyse no wash flow cytometry for maximizing minimal sample preparation. Methods. 134-135: 149-163.
  7. Nandakumar S, Grushko O, Buttitta LA (2020) Polyploidy in the adult Drosophila brain Elife. Aug 25 (9) e54385.
  1. Rangel GW, Clark MA, Kanjee U, et al. (2020) Plasmodium vivax transcriptional profiling of low input cryopreserved isolates through the intraerythrocytic development cycle. PLoS Negl Trop Dis 14(3): e0008104.
  2. Bourge M, Brown SC, Siljak-Yakovlev S. (2018) Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment. Genetics & Applications, [S.l.], v. 2, n. 2, p. 1-12, dec. 2018. ISSN 2566-431X.
  3. Broughton KM, Khieu T, Nguyen N, et al. (2019) Cardiac interstitial tetraploid cells can escape replicative senescence in rodents but not large mammals. Commun Biol 2, 205.
  1. Gaydosik AM, Queen DS, Trager MH, et al. (2020) Genome-wide transcriptome analysis of the STAT6-regulated genes in advanced-stage cutaneous T-cell lymphoma. Blood, 136 (15), 1748-1759.
  2. Bernlochner I, Klug M, Larasati D, et al. (2020) Sorting and magnetic-based isolation of reticulated platelets from peripheral blood. Platelets, Feb 11:1-7.
  3. Benazic S, Silconi ZB, Jevtovic A, et al. (2020) The Zn(S-pr-thiosal)2 complex attenuates murine breast cancer growth by inducing apoptosis and G1/S cell cycle arrest. Future Med Chem 2020 12:10, 897-914.

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