The cell cycle is the process by which eukaryotic cells duplicate and divide. The cell cycle consists of two specific and distinct phases: interphase, consisting of G1 (Gap 1), S (synthesis), and G2 (Gap 2), and the mitotic phase; M (mitosis) (Figure 1). During interphase, the cell grows (G1), accumulates the energy necessary for duplication, replicates cellular DNA (S), and prepares to divide (G2) [2]. At this point, the cell enters the M phase, which is divided into two tightly regulated stages: mitosis and cytokinesis. During mitosis, a parent cell's chromosomes are divided between two sister cells. In cytokinesis, the division of the cytoplasm occurs, leading to the formation of two distinct daughter cells. Each phase of the cell cycle is tightly regulated, and checkpoints exist to detect potential DNA damage and allow it to be repaired before a cell divides. If the damage cannot be repaired, a cell becomes targeted for apoptosis. Cells can also reversibly stop dividing and temporarily enter a quiescent or senescent state; G0. The first checkpoint is at the end of G1, making the decision if a cell should enter S phase and divide, delay division, or enter G0. The second checkpoint, at the end of G2, triggers mitosis if a cell has all the necessary components.

[enlarge]

Figure 1. Schematic of the cell cycle; Image provided courtesy of Abcam Inc. Copyright©2013 Abcam

Several methods to assess the cell cycle are discussed below. However, it is important to remember that these methods are not mutually exclusive, and for the best and most reliable data multiple dyes and/or analytes can be combined in a single experiment or multiple assays used.

[enlarge]

Figure 2. Method for assessing the cell cycle by FACS

The most common method for assessing the cell cycle is to use flow cytometry to measure cellular DNA content. During this process, a fluorescent dye that binds to DNA is incubated with a single cell suspension of permeabilized or fixed cells. Since the dye binds to DNA stoichiometrically, the amount of fluorescent signal is directly proportional to the amount of DNA. Because of the alterations that occur during the cell cycle, analysis of DNA content allows discrimination between G1, S, G2 and M phases. The simple protocol for cellular analysis is outlined in Figure 2. Briefly, cells are fixed and permeabilized to allow the dye(s) to enter the cell and to prevent them from being exported out. Staining with the DNA binding dye then occurs after cells have been treated with RNase to ensure only DNA is being measured. Several datasets, including forward scatter vs. side scatter, pulse area vs. pulse width, and cell count vs. propidium iodide, are collected to ensure only single cells are measured. Examples of these traces are shown in Figure 3.

[enlarge]

Figure 3. Characteristic traces of cell cycle FACS analysis. Images provided courtesy of Abcam Inc. Copyright©2013 Abcam

There are several different dyes that can be used in these assays, including propidium iodide (PI) [3, 4], 7-amino actinomycin-D (7-AAD), Hoechst 33342 and 33258, and 4’6’-diamidino-2-phenylindole (DAPI). For example, Chopra S et al labeled mouse bone marrow–derived dendritic cells and paw single cell suspensions with 0.5 μg/ml DAPI from Thermo Fisher during flow cytometry on a BD LSR II instrument and cell sorting with a BD Aria II SORP cell sorter [5]. Zhang H et al combined mitosis-specific anti-pMPM2 antibody ( 05-368 from MilliporeSigma) staining with DAPI to obtain prometaphase cells through FACS [6]. However, most FACS machines commonly used contain only single argon-ion lasers, and as such dyes requiring UV activation such as DAPI and Hoescht 33342 are less frequently used. A derivative of Hoechst dye, SiR-Hoechst, has excitation at 640 nm, and thus may find widespread use [7]. Hoechst 33258 has also been used to image polycomb bodies [8]. Rhodes JDP et al estimated the proportion of mitotic cells through antibody staining of serine 10 phosphorylated histone H3 in FACS [8]. Phosphorylated histone H3 staining has also been used in immunohistochemistry to identify mitotic cells in mouse [9, 10] and killifish [11].

When carrying out these analyses, it is important to recognize that simple single stained FACS analysis using 7-AAD or PI is unable to distinguish between cells in G1 or G2 from those in very early or very late S phase, and similarly those in G2 or M. It is therefore sometimes necessary to combine these dyes with a proliferative marker such as BrdU [3]. For example, Calvanese V et al combined 7AAD and BrdU-PE staining in flow cytometry to assess the cell cycle stages of cultured hematopoietic stem or progenitor cells [12]. This requires additional steps at the beginning of the study, where live cells still in culture are incubated with BrdU for a period of approximately 30 minutes (or 2 hours for immunocytochemistry analysis [13] ) , before incubation with anti-BrdU and fluorescence-conjugated secondary antibodies. Cells are then assayed as described above.

There are a number of important considerations when carrying out analysis of cell cycle FACS data. The forward scatter/side scatter plots are an integral part of the analysis and should not be overlooked, since this is how single cells are identified. If doublets (when the DNA content of two cells in G1 are recorded as a single G2/M event) are allowed in the analysis, it can lead to over-representation of G2/M. Cellular aggregates and flow rates below 1000 cells/second should also be avoided to allow a low sample pressure differential to be used, which leads to an optimal coefficient of variance (CV). Finally, reference samples containing normal diploid DNA should be included as an additional control.

The Nicoletti assay [14] is a modified form of cell cycle FACS analysis that concurrently allows apoptosis to be assessed by measuring cells with low intact DNA content, and high fragmented DNA content [15] (the pre-G1 peak). The Nicoletti method is very similar to that described above, with the exception that a hypotonic buffer (such as HFS buffer containing sodium citrate and Triton X-100, or a hypotonic fluorochrome solution) is used to permeabilize the cells. Apoptotic cells stain weaker in these assays due to the activation of cellular nucleases and the diffusion of low molecular weight DNA out of the cell. Fixing and permeabilizing cells stimulates the release of oligo- and mononucleosomes. The use of a hypotonic buffer facilitates the loss of fragmented DNA, resulting in a shift of the pre-G1 peak.

The images in Figure 4 demonstrate healthy cells (top), cells in which a sub-population is beginning to undergo apoptosis (middle), and a population of cells with extensive apoptosis (bottom). However when using the Nicoletti assay, care must be taken to discriminate apoptotic nuclei from cell debris, and to ensure that DNA shearing does not occur during the fixing and staining processes.

[enlarge]

Figure 4. Nicoletti FACS traces

The cyclins are key regulatory components of the cell cycle machinery. The cyclin family comprises the classical cyclins, cyclin-dependent kinases [16, 17] (CDKs) and Cdk inhibitors (CKIs). Although there is much redundancy between the individual cyclins and CDKs [18], the activity and expression of the individual proteins fluctuate during each distinct phase of the cell cycle, playing an important regulatory role. Although this is a complex and highly regulated process, in general cyclins can be divided into sub-groups governed by the phase of the cell cycle they regulate, summarized in Figure 5. For example, Cyclin D1 is required for the passage of cells from G0 to G1. Once expressed, it forms a complex with Cdk4, which activates retinoblastoma protein, leading to the upregulation of Cyclin E. Cyclin E, in combination with cyclin A, then interacts with Cdk2 to promote G1/S transition. In contrast, cyclins B1 and B2 are expressed during M phase where they interact with Cdk1 to form part of the MPF (M phase/maturation promoting factor), an assembly that regulates a cascade of processes leading to mitotic spindle assembly and ultimately cell division. The expression of each human cyclin and their interaction with Cdks are summarized in table 1.

These distinct expression patterns can therefore be exploited during cell cycle analysis. The total levels and/or phosphorylation status of individual cyclins can be easily and rapidly measured using specific antibodies by immunoblotting [3]. In addition, specific ELISA kits are available for individual cyclin family components, allowing for a more quantitative assessment of expression. Finally, fluorescently conjugated antibodies can be used in immunohisto- or immunocyto-chemical approaches, or in flow cytometry. Combining cyclin staining with FACS methods examining DNA content provides a powerful and quantitative tool to analyze the cell cycle accurately [3].

[enlarge]

Figure 5. Differential expression of cyclins during the cell cycle

Tetraploid cells are associated with the formation of malignancy and often possess the stem-cell characteristics. Thus, with relevance to cancer biology [19, 20] and regenerative tissue homeostasis [21], it is conceivable that the analysis of tetraploid cells would be of importance. The tetraploid G1 cells and diploid G2/M cells are difficult to detect as they possess the same ploidy that is 4C DNA content.

FUCCI (Fluorescence ubiquitination-based cell cycle indicator) system is a technology that utilizes the cell cycle phase-specific expression of proteins and their degradation by the ubiquitin-proteasome degradation system [22, 23]. The technology analyzes the living cells in a spatio-temporal manner using dual-color protein-fluorescent chimeras. Moreover, it enables to overcome the problem of isolating the cells in different phases, which is otherwise difficult to differentiate only with the DNA-based stains such Hoechst. It is composed of two proteins - Cdt1 (Cdc10 dependent transcript 1) and Geminin. Both proteins are used in their truncated forms (hCdt1 and hGeminin) and are conjugated to two different fluorescent proteins. They express alternately in the two different cell cycle phases. Cdt1 is a conserved replication factor required for licensing the chromosome for DNA synthesis. Cdt1 is expressed throughout the G1 phase and is ubiquitinated by the ubiquitin ligase complex SCFSkp2 during S and G2/M phases followed by its degradation by the proteasome. In contrast, geminin inhibits the licensing activity of Cdt1 by interfering with the binding of licensing factors to the replication origin during the S phase. It is present during S/G2/M phases. At the end of M phase and throughout the G1 phase, geminin is ubiquitinated by the E3 ligase complex APCcdh1 and degraded by the proteasome [22].

Figure 6 depicts the scatterplot representing the live cells expressing different fluorochromes implying their diverse cell cycle phases. Depending on the probe selection, the two chimeras emit different fluorescence. Several probes are available commercially. One of the examples is stated below along with a diagram. Fucci-G1 Red is a fusion protein of a fragment of human Cdt1 (amino acids 30-120) with the red fluorescent mCherry-RFP, that detects the cells in G1 phase. Fucci-S/G2/M Green is a fusion protein of a fragment of human geminin (amino acids 1-110) with the green fluorescent protein mAG1 (monomeric Azami-Green1) that visualizes S, G2 and M phases. Thus, the G1 tetraploids emit red fluorescence and G2/M tetraploids emit green fluorescence. By employing this technology, it is also possible to distinguish between the mononucleated diploid cells from the binucleated cells.

[enlarge]

Figure 6. (A) Histogram displaying 2C and 4C DNA content by the Hoechst 33342 stain. (B) Scatterplot representing the G1 (orange) and G2/M diploids (green) cells expressing two different fluorescent proteins. (Image adapted from [1] ).

Table 2 lists some of the commercial suppliers of FUCCI kits. The FUCCI sensors from Thermo Fischer Scientific, for example, were used to analyze G1 phase in mouse stem cells and evaluate mechanisms of UV-mediated damage [25] and investigate the awakening and proliferation of dormant metastatic cells by neutrophil extracellular networks [26]. M Barnat et al obtained pCAG-Geminin-GFP and pCAGCdt1-mKO2 from A. Miyawaki, RIKEN Brain Science Institute, Japan [10]. L Crozier et al utilized a FUCCI cell line to investigate the effect of CDK4/6 inhibitors on cell cycle progression [27].

One of the limitation of the FUCCI system is that these systems requires the expression of multiple reporter constructs intracellularly and reduces the chance to image other targets spectrally. This problem has been overcome by modification of this system. Zerjatke et al developed fluorescently tagged endogenous proliferating cell nuclear antigen (PCNA) as an all-in-one cell cycle reporter. This reporter with PCNA-mRuby alters in brightness and localization in different phases. Consequently, it provides a readout of the cell cycle phase including quiescence and quantitative dynamics of individual fate determinants of cell cycle regulation [28].

Another limitation is that FUCCI system and its variants allow visualizing whether cells are within one of the proliferative phases (S, G2, or M) of the cell cycle, they do not report simultaneous visualization of the three phases cells in real time. Bajar et al developed a robust method that enables simultaneous imaging of the all four phases. They established an intensiometric reporter for the S/G2 transition and further engineered a far-red fluorescent protein, mMaroon1, to track the process of mitotic chromatin condensation. They designed a new version called Fucci4 by combining the new reporters with the FUCCI system and incorporating four orthogonal fluorescent indicators that enable to capture all cell cycle phases in the living cells [29]. Fucci4 has diverse applications in development, physiology, and cancer. Fucci4 allows 1) how diverse changes at the molecular, genetic and extracellular signaling level alter the cell cycle, 2) molecular mechanisms regulating specific phase transitions and 3) screening for drugs that affect a particular cell cycle phase or cell cycle distribution.

There are several applications of FUCCI system in various branches of biology and medicine. For studying development biology, Sugiyama et al generated transgenic Zebrafish lines expressing the non-mammalian FUCCI counterparts [30]. They were employed to study the spatio-temporal regulation of cell-cycle progression during major morphogenetic events/processes (gastrulation, metamorphosis, involution, invagination and branching) [22, 31]. Zielke et al engineered Drosophila-specific FUCCI system (Fly-FUCCI) that involves tissue-specific expression of the FUCCI probes [32]. This allows one to distinguish G1, S, and G2 phases of interphase. This serves as a valuable tool for visualizing cell-cycle activity during development, tissue homeostasis, and neoplastic growth.

The FUCCI system can be used in tumors for in vivo cell cycle profiling by stably transfecting cell lines with FUCCI reporters and development of the xenograft tumors [33]. Nico Battich et al correlated the synthesis and degradation rates of mRNAs along the cell cycle indicated by the FUCCI system [34].

Sawano et al re-engineered the Cdt1-based sensor from the original Fucci system to respond to S phase-specific CUL4Ddb1-mediated ubiquitination alone or in combination with SCFSkp2-mediated ubiquitylation. This system is known as Fucci(CA) and it demarcates interphase with boundaries between G1, S, and G2. The applications of Fucci(CA) included tracking the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying UV-irradiation damage in S phase [25].

Several new reagents for cell cycle analysis, such as chromobodies and Cycletest reagent, have recently been developed. Chromobodies are fusion proteins, which contain fluoresceins linked to the antigen binding domain of heavy chain antibodies. These reagents are used to detect the expression of various intracellular proteins within the cellular compartments and dynamic changes of their distribution during different phases of the cell cycle. Furthermore, chromobodies can be applied for the detection of both cytoskeletal and nuclear proteins. With regard to cytoskeletal target proteins, changes in vimentin expression have been analyzed by specific chromobodies in a study that generated vimentin knock-out cell line [35]. As to the analysis of nuclear proteins by chromobodies, Proliferating Cell Nuclear Antigen (PCNA), which plays a crucial part in the replication process in the nucleus, was detected by the red fluorescent protein-bound chromobody [36]. Also, an advanced modification of PCNA detection by chromobodies, which was based on 4D quantitative analysis of the PCNA expression and distribution during the replication phase, has recently been reported [37].

Moreover, chromobody assays can be combined with other techniques. For example, a combination of chromobody-based analysis of the cell cycle with the Chto Tox-Glo cytotoxicity method has been described. In that study, the visualization of PCNA expression in subcellular compartments by chromobodies was followed by the evaluation of protease activity in vitro [38]. With regard to the applications of chromobodies for in vivo research, the chromobody-based method has been originally applied to studies in zebrafish [39]. In addition, Wegner et al have analyzed the expression of actin in the mouse brain using anti-actin chromobodies labelled with fluorescent protein mNeptune2. [40].

In addition, Cycletest PLUS reagent kit produced by BD Biosciences is recommended for cell cycle analysis. This method includes the elimination of the cell membrane and cytoskeleton with a detergent and trypsin, respectively, followed by the digestion of RNA and stabilization of the chromatin. The kit contains propidium iodide (PI), which binds to the extracted nuclei. The stained nuclei are analyzed by flow cytometry to measure the binding of PI to DNA. This reagent was successfully used in several recent studies. For instance, Cycletest has been applied to evaluate the anti-tumor effects of thymoquinone in human breast tumor MCF-7 cells [47] and the effects of α-solanine in colorectal tumor cells [48].

This section is provided by Labome to help guide researchers to identify most suited cell cycle analysis assay kits. Labome surveys formal publications. Table 3 lists the major suppliers for reagents/kits used in the cell-based assays and their numbers of publications in the Labome survey. The review article on cell proliferation lists the survey results on BrDU.

This article is derived from an earlier version of an article authored by Dr. Laura Cobb "Cell-Based Assays: the Cell Cycle, Cell Proliferation and Cell Death", written in February 2013. Dr. Samayita Das contributed to the section on the FUCCI system in September 2019.