There are different types of cell cycle analysis assays, and the method you choose depends on if you want to use flow cytometry or imaging and if you need fixed or live cell cycle analysis.
Flow Cytometry—Get the percentages of a cell population in the different phases of the cell cycle, in live or fixed cells
Microscopy—Visualize live cell division within a population
Antibodies—Study cell cycle dysregulation
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Cell cycle analysis by assessing DNA content distribution
The eukaryotic cell cycle, or cell division cycle, compromises a series of events that are involved in the growth, replication, and division of cells. The cell cycle describes the progression of a cell through a cycle of division (Figure 1) and involves both cytoplasmic and nuclear events.
- G1 phase (growth 1 phase)—cells induce growth by first making RNA and proteins, DNA content does not change
- S phase (synthesis phase)—DNA synthesis proceeds with DNA replication and cells have variable amounts of DNA
- G2 phase (growth 2 phase)—have completed DNA synthesis, and continue to grow and prepare for mitosis with DNA maintained at double the original amount
- M phase (mitosis)—nuclear division and cytoplasmic division giving rise to two daughter cells
Figure 1. Phases of the cell cycle. Phases G1, S, and G2 comprise interphases of the cell cycle. M (mitosis) comprises 4 distinct phases: prophase, metaphase, anaphase, and telophase. G0 is the resting phase where cells exist in a quiescent, non-dividing state.
After completion of the M phase (mitosis), each daughter cell contains the same genetic material as the original parent and approximately half of its G2 level of cytoplasm. Cells can exit from the cell cycle and enter a non-dividing stage (G0), and once there, they can enter the cycle with proper activation.
Cells can be incubated with dyes that bind stoichiometrically to DNA. This means that the dye binds in proportion to the amount of DNA present in each cell. Using flow cytometry, a single time point measurement can be recorded for the DNA content distribution within a cell population. With linear fluorescence and stoichiometric staining, the fluorescence of the 4N cells is expected to be exactly twice the 2N cells (Figure 2A). In practice, the cell populations are represented on frequency histograms with peaks of various widths. Variations result from differences in staining, dye loading, instrumentation, and the data are similar to but not exactly like the model. Shown below is an example of commonly observed cell cycle data that was obtained with Invitrogen Vybrant DyeCycle Violet staining (Figure 2B).
Figure 2. Example DNA content distribution in a flow cytometry cell cycle analysis assay. (A) Not actual data, this plot demonstrates the DNA content of cells in different phases of the cell cycle and how to interpret a DNA content histogram. Brown circles show where G0 and G1 cells would be, both having 2X DNA. Yellow circles show where S phase cells with varying and increasing DNA content would be expected to be observed. The red circle show where M phase cells with 4X DNA before division would be found. Blue circles demonstrate where G2 phase cells also with 4X DNA, would be observed.
The fluorescence of the 4N cells at G2M is expected to be exactly twice the 2N cells at G0/G1. (B) In reality, what is observed is variation in peak widths in the frequency histograms of the cell populations. These variations are expected and are attributed to multiple sources of variations within an experiment.
Detection of senescent cells
When normal cells reach the end of their limited replicative lifespan, commonly referred to as the Hayflick limit, they enter a senescent phase where they remain metabolically active without undergoing cell death processes. These senescent cells adopt a specific phenotypic state that includes the appearance of multi-nucleated cells, increased vacuolization, expression of pH-dependent β-galactosidase, and morphological changes where cells become enlarged and extended. Through a variety of mechanisms, they may play a role in tumor suppression, tumor progression, aging, and tissue repair.
Activation of β-galactosidase is commonly used as a biomarker for senescent cells (Figure 3A and 3C). This hydrolase enzyme resides in lysosomes and converts β-galactosides into monosaccharides under acidic pH conditions. Traditionally, the colorimetric substrate for β-galactosidase, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), has been used to detect metabolic activity in cells in vitro in imaging applications, but this assay is not suitable for use in flow cytometry. The CellEvent Senescence Green Probe is a fluorescent-based reagent that provides greater sensitivity than traditional colorimetric X-gal in imaging applications and is also amenable to flow cytometry applications (Figure 3B and 3D).
Figure 3. Senescent and non-senescent cells treated with CellEvent Senescence Green Probe or X-gal. T47D cells treated with 5 µM palbociclib via media changes every other day for 15 days to induce senescence through cyclin D checkpoint blockade (senescent) and untreated T47D cells (non-senescent) were fixed in 4% paraformaldehyde for 10 minutes at room temperature, then stained with either CellEvent Senescence Green Probe or X-gal for 90 minutes in a 37°C incubator with no CO2. Panels A and C were stained with X-gal; Panels B and D were stained with the CellEvent Senescence Green Probe.
Figure 4. Observed change in expression of cyclin A2 and B1 in senescent and non-senescent cells as analyzed using the CellEvent Senescence Green Flow Cytometry Assay. Cyclin A2 and B1 expression decreases with onset of senescence.
Commonly used stains for cell cycle analysis assays by flow cytometry
Flow cytometry, in conjunction with modeling algorithms, provide a powerful tool to assess cells in G0/G1 phase versus S phase, G2, or polyploidy. We offer classic DNA cell cycle stains such as Hoechst 33342, DRAQ5, DAPI, and PI along with a series of improved fluorescent dyes, the Invitrogen FxCycle reagents for fixed cell cycle analysis, and the Invitrogen Vybrant DyeCycle reagents for live cell cycle analysis. These improved reagents allow accurate cell cycle analysis with more color options. Additionally, the Vybrant DyeCycle reagents typically exhibit such low toxicity that stained cells can be sorted and otherwise cultured or assessed.
Live cell imaging of cell cycle and division
Visualize cell division within a cell population using a genetically-encoded, two-color (red and green) indicator, the Invitrogen Premo FUCCI Cell Cycle Sensor (Figure 4). This sensor can be used to efficiently label and follow cells during their division using fluorescence microscopy.
Antibodies for cell cycle analysis research
The cell cycle is a regulated series of molecular events that dictates cellular division and proliferation. G1, S, G2 (collectively termed ‘interphase’), and M (mitotic) phases are the major stages of the cell cycle. Cyclins and cyclin-dependent kinases (CDKs) play a vital role in orchestrating the events pertaining to each of these stages. The expression of cyclins varies throughout the cell cycle as they bind to and activate specific CDKs. CDK activity is regulated by CDK inhibitors belonging to two major families: INK4 proteins, including INK4A (p16), INK4B (p15), INK4C (p18), and INK4D (p19), and the Cip/Kip family, which includes p21 (Cip1), p27 (Kip1), and p57 (Kip2). Dysregulation of cell cycle is implicated in the development of various diseases including cancer.
The availability of high-quality antibodies is crucial to study the key molecular players of cell cycle in various research applications. For example, Aurora B is a component of the chromosomal passenger complex (CPC) and functions in centrosome duplication, chromosome alignment and separation, histone modification, and cytokinesis. The overexpression of Aurora B has been reported in various tumor types and is also associated with poor prognosis in cancer patients. Figure 5 shows the basal expression of Aurora B across various cell lines and its upregulation upon Nocodazole treatment in HeLa cells using Aurora B Recombinant Rabbit Monoclonal antibody (RM278) (Cat. No. MA5-27890) in western blot application. Aurora B localizes at centromeres during prometaphase and relocates to midzone microtubules and midbodies during anaphase and telophase. Figure 6 shows the dynamic localization of Aurora B as observed in immunofluorescence application using the same antibody.
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Figure 5. Aurora B Antibody in a Western blot. Western blot was performed using Anti-Aurora B Recombinant Rabbit Monoclonal Antibody (RM278) (Cat. No. MA5-27890), and a 39 kDa band corresponding to Aurora B was observed across cell lines and tissue tested.
Figure 6. Aurora B Antibody in an immunofluorescence experiment. Immunofluorescence analysis of A549 cells labelled with Aurora B Recombinant Rabbit Monoclonal Antibody (RM278) (Cat. No. MA5-27890).
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