Flow cytometry is the technique of choice for analyzing the cell cycle. This article comments on how the methodology has evolved and shares some strategies that can help simplify the workflow.
Flow cytometry is fundamental to cell cycle analysis
Researchers study the cell cycle to assess cellular health, genotoxicity, and drug efficacy, as well as to understand how cell cycle dysregulation is linked to conditions such as cancer, atherosclerosis, and Alzheimer’s disease. Cell cycle knowledge can also be important for optimizing the production of biopharmaceuticals in bioreactors and enhancing biomass and yield in major crops. “Flow cytometry is fundamental to these applications as it allows researchers to rapidly, precisely, and reproducibly determine the different phases of the cell cycle,” reports Dr. Pia Jeggle, Group Leader, Flow Cytometry Instruments at Miltenyi Biotec. “Such speed, reliability, and ubiquitous accessibility, combined with the flexibility for the researcher to tailor the experiment to their specific needs, is currently not fulfilled by other methodologies.”
DNA content analysis provides only limited information
Cell cycle analysis has traditionally involved flow cytometric evaluation of cellular DNA content using dyes such as propidium iodide (PI), 7-AAD, Hoechst 33342, and DAPI to determine the proportion of cells within each stage of the cell cycle. “These dyes bind DNA in a stoichiometric manner, so cells in G1 phase will have half as much DNA as cells in G2, which is reflected in the measured fluorescence,” explains Dr. Christopher Brampton, Global Product Manager for Applications and Flow Reagents at Bio-Rad Laboratories.
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“However, a drawback of this approach is that it provides only limited information. For example, it is impossible using DNA content alone to distinguish quiescent/G0 cells from G1, differentiate between G2 and M, or accurately determine the percentage of cells in S phase. For this reason, researchers usually combine DNA content analysis with the detection of cell cycle markers for a more detailed picture of what cell cycle processes or pathways are changing in each cell. This can especially be useful for drug development programs aimed at targeting a particular facet of the cell cycle.”
Cell cycle markers offer deeper insights
Kamala Tyagarajan, Ph.D., Director, Flow Cytometry Assays and Applications at Luminex, notes that monitoring the incorporation of thymidine analogs such as BrdU into newly synthesized DNA, followed by detection with anti-BrdU antibodies and PI, is an established method for better teasing out cells in S phase. “Other popular antibody-based strategies include staining for nuclear antigen Ki67, a marker of actively cycling proliferating cells that is thought to be degraded in G0 phase; assessing levels of proliferating cell nuclear antigen to differentiate between S phase sub-stages; and detecting phosphorylated histone H3, which is a marker of M phase,” she says. “Additionally, analyzing peak expression of cyclins and cyclin-dependent kinases along with DNA content analysis is helpful in characterizing different cell cycle phases.”
Complementing these methods, Thermo Fisher Scientific’s Invitrogen™ Click-iT™ Plus EdU cell proliferation assays were developed as non-antibody-based alternatives to using BrdU that circumvent the need for DNA denaturation. “Detecting BrdU requires DNA denaturation using acid, heat, or nuclease treatment, which can introduce unwanted artifacts and also prolongs the time to detection,” reports Jolene A. Bradford, Senior Product Manager at Thermo Fisher Scientific. “EdU is instead detected via a click reaction—a type of reaction that proceeds quickly and selectively under mild conditions to covalently link specific molecular components, in this case EdU and a fluorophore-labeled picolyl azide for direct measurement of cells in S phase.”
Click-iT™ Plus EdU cell proliferation assays employ a simple, robust protocol and are compatible with most standard fluorophore conjugates.
Addressing challenges of flow cytometry-based cell cycle analysis
Flow cytometry-based cell cycle analysis comes with its own challenges. Critically, the presence of doublets and clumps can interfere with determining ploidy and generating reliable results. “Proper sample preparation is crucial for cell cycle analysis and should include steps aimed at ensuring a single-cell suspension,” comments Jeggle. “Clumping can be prevented by avoiding overly harsh centrifugations, adding DNase to samples, and using resuspension buffers that are free from calcium and magnesium cations. Passing cells through a cell strainer is also recommended, while gating out doublets as well as dead cells will help improve data quality.”
A further common problem faced by researchers performing more in-depth cell cycle analysis is that it requires the cells to be fixed, reducing the potential for other downstream applications. “Cell cycle analysis is often performed on cells fixed in 70% ethanol at -20oC, which can have detrimental effects on other stains and markers you may be interested in,” says Brampton. “Hoechst is an exception to this as it has low toxicity and readily crosses the cell membrane to bind DNA, making it useful for live cell sorting.” Another option for labeling living cells is to use cell-permeant DNA dyes such as Thermo Fisher Scientific’s Invitrogen™ Vybrant™ DyeCycle™ stains, which can be excited with a range of different lasers, providing increased flexibility for multiplexing.
Extending the scope of cell cycle analysis
Other technologies developed to streamline cell cycle analysis include next-generation versions of existing DNA dyes that reduce the number of handling steps for more reproducible results. For example, Bio-Rad’s PureBlu™ Hoechst 33342 eliminates the need for a weighing step and requires only a single dilution after resuspension, while ReadiDrop™ Propidium Iodide can be used neat and necessitates just a 20 minute incubation with the sample. The latter, used in combination with Click-iT™ Plus EdU staining, is cited in a recent publication, where researchers generated a reporter line for PAX7 in human induced pluripotent stem cells. In this study, PAX7+ cells were shown to produce myofibers and self-renew in vitro and in vivo, a finding with significance for treating muscular dystrophy.
Advances in instrumentation have also helped drive novel discoveries. Using Miltenyi Biotec’s MACSQuant flow cytometers, which pair automatic sample labeling with automatic volumetric cell counting, researchers have found that Myc stimulates cell cycle progression through the activation of Cdk1 and phosphorylation of p27; shown that CRK3 has an essential role in the cell cycle of Leishmania mexicana, suggesting it to be an important therapeutic target for tackling leishmaniasis, a disease that causes an estimated 40,000 deaths annually; and proven that chemoresistance in cancer stem-like cells is related to the cell cycle.
Platforms leveraging newer technologies such as imaging flow cytometry and artificial intelligence (AI) are likewise advancing researchers’ understanding of the cell cycle. For example, Luminex’ Amnis® ImageStream®X and Flow Sight® imaging flow cytometers combine fluorescent staining for nuclear/cell cycle markers along with morphological data and further analysis with AI for more detailed information about different cell cycle stages. “This approach maintains the advantage of sampling a large number of cells while providing more powerful integrated data,” explains Tyagarajan. “As efforts to elucidate the mechanisms of the cell cycle continue, methods such as imaging flow cytometry along with deep learning will provide enriched information on the stages of cell cycle pivotal to future breakthroughs.”