The cell cycle is a tightly coordinated process that directly impacts the growth, development, and replication of all eukaryotic cells. Divided into four main phases, it combines DNA replication and chromosomal segregation in an oscillatory manner to ensure that upon division each resulting daughter cell receives an equal complement of genetic material. Dysregulation of the cell cycle is implicated in many disease states, most notably cancer, therefore the ability to accurately monitor cell cycle progression is essential to helping researchers better understand the various mechanisms underlying its control.
“The simplest approach to monitoring the cell cycle is to assess proliferation by counting cells,” explains Paul Wylie, head of applications at TTP Labtech, “and the most common of way of achieving this is to determine DNA content using cheap dyes and simple analysis techniques such as flow cytometry or microscopy. Yet while these methods are highly informative and can be used to identify cells within each stage of the cell cycle, they represent a global view of the process. To study individual phases in more detail, researchers may choose to incorporate immunostaining protocols that use commercially available antibodies to target the cyclical expression of specific proteins.”
“Flow cytometry is notoriously prone to clogging, a factor that significantly reduces its applicability to automation and which can disrupt screening runs,” adds Sarah Payne, product manager. “Yet while a microscope-based approach represents a viable alternative to flow, it can pose additional challenges and bottlenecks due to the mountains of data that are produced and the requirement for specialist operators. To address these issues, we developed our acumen® Cellista to bring the benefits of cytometry to the screening environment.”
According to Wylie, this powerful instrument employs an innovative laser scanning image-cytometry approach. “Acumen® Cellista can read and analyze the cell cycle phase of all cells in a 1,536 well microplate in just five minutes,” he says, “allowing for throughput of up to 150,000 compounds per day. Adherent cells are analyzed in situ 
to preserve their morphology, with results normalized to total cell number highlighting proliferation, cytotoxicity, or cytostasis, while the fluorescent DNA binding dyes used to quantify cell cycle phases may be re-analyzed over time to monitor kinetics. In addition to rapid multiparameter analysis, acumen Cellista affords multiplexing using immunofluorescence protocols to profile the expression of different cell cycle markers.”
Image: Concentration-dependence of vinblastine treatment on the cell cycle, measured using acumen® Cellista.
“Many of the techniques currently used to monitor the cell cycle, such as BrdU incorporation, flow cytometric analysis of propidium iodide (PI) stained cells, or immunostaining of cell cycle markers, require cell fixation prior to analysis,” notes Christoph Eckert, marketing and sales manager at ChromoTek. “In contrast, our Cell Cycle Chromobody® (CCC) empowers researchers to resolve dynamic changes within live cells in real time by highlighting an endogenous cell cycle marker protein, proliferating cell nuclear antigen (PCNA). Although over-expression of PCNA has conventionally been used to monitor cycling cells, CCC does not change endogenous PCNA levels and therefore has no effect on cell cycle progression.”
The CCC signal varies throughout the cell cycle, with homogeneous 
distribution throughout the nucleus and cytoplasm during G1, and nuclear accumulation within S phase. This visualizes the formation of replication foci, which disappear in G2 prior to cell division.
Image: Cell Cycle Chromobody® signal during the cell cycle.
“To complement CCC, our Chromobody products can easily be combined for correlative studies,” adds Eckert. “For instance, by co-transfecting cells with two plasmids that encode these extremely small intracellular functional antibodies carrying distinct fluorophores, simultaneous detection of different cell cycle proteins and cellular markers is possible.” This is exemplified in the 2015 publication by Panza et al, which includes the use of chromobodies for live imaging of endogenous protein dynamics in zebrafish.
Another reagent designed primarily for live cell imaging of cell cycle progression is Thermo Fisher Scientific’s Premo™ FUCCI Cell Cycle Sensor. This is based on two cell cycle-regulated proteins, geminin and Cdt1, fused to green and red fluorescent proteins, respectively. Germinin and Cdt1 are each degraded within different phases of the cell cycle, with both being present during the G1/S transition. This produces a dynamic color change from red to yellow to green. “The Premo FUCCI Cell Cycle Sensor is delivered by BacMam 2.0 technology,” explains Brian Almond, senior manager, product development. “This employs a VSVG-pseudo-typed capsid protein for efficient cell entry, a strong mammalian promoter, and a post-transcriptional regulatory element that results in higher expression levels. These features make the reagent suitable for cell cycle studies in essentially any cell type, including primary neurons, stem cells, and cardiomyocytes, all of which can be difficult to transfect.”
Cells labeled with Premo FUCCI Cell Cycle Sensor can also be stained with antibodies to other cellular targets, despite the reagent having been developed for live cell imaging. “The fluorescence from geminin-GFP and Cdt1-RFP have been demonstrated to be resistant to fixation with 4% formaldehyde and permeabilization with 0.1% Triton® X-100,” says Almond, “and for further confirmation of cell cycle phase you can accurately correlate expression of the reagent with DNA content using spectrally compatible blue stains such as Hoechst 33342.”
In addition to an extensive portfolio of DNA dyes and antibodies against key cell cycle markers such as cyclins, retinoblastoma, and phosphorylated histone H3, BD Biosciences offers the Cycletest™ Plus reagent kit for quick and accurate analysis of cellular DNA content via flow cytometry. “Cycletest Plus™ provides an optimized set of reagents for isolating and staining cell nuclei from fresh or frozen solid tissue specimens or cell suspensions, making it suitable both for in vitro and in vivo studies,” explains Matthew Miceli, global product manager, research reagents.
“Based on propidium iodide, well-known to exhibit stoichiometric DNA staining, specific reagents within the kit degrade proteins and RNA to allow more precise DNA measurement. This product has been widely cited in the literature for studies reliant on cell cycle monitoring. For example, a 2017 publication in the Journal of Experimental and Clinical Cancer Research details the kit’s use in research to detect cell cycle arrest following treatment of gastric carcinoma cell lines with the small molecule ICG-001.”
Although researchers continue to use tried-and-trusted methods to monitor the cell cycle, companies are developing increasingly sophisticated means of achieving this analysis. Almond concludes that “not only are mechanisms to track and identify cell cycle progression useful for oncology researchers to determine which phase cells are in and if they are progressing normally through the cell cycle, but the phase of the cell cycle can determine how a cancer cell will respond to a therapeutic drug. Stem cell researchers also rely on tools to monitor the cell cycle, for example within regenerative studies for organ growth, in which the aim is to switch dormant cells back to replicative cells. Yet another area of active research focuses on how the cell cycle responds to DNA damage. DNA repair operates throughout the entire cell cycle but is especially important in G1. Knowing which phase the cells are in is key to studying the cellular mechanisms of repairing damaged DNA.”