Disease states such as cancer arise from an imbalanced or uncontrolled division of cells in the body. Being able to monitor cell cycle progression and flag abnormalities is key to understanding not only cancer but states such as senescence and cellular aging.

How their cycle is disrupted in disease, as well as how it responds to interventions like drug treatment, hypoxia, transgenesis, and radiation, for example, can give robust insights into health, disease, and the mechanisms of action of such treatments. Such knowledge is not limited to humans, either: understanding, predicting, and ultimately controlling the cell cycle in such lowly organisms as yeast and bacteria are paramount in bioprocess, for example.

Since its inception a half-century ago, flow cytometry has afforded researchers a powerful means to determine the phase of the cell cycle, alongside other relevant parameters, of thousands of individual cells simultaneously. Here we look at how flow is used to monitor the cell cycle, from simple single-color DNA content analysis to multiparameter immunophenotyping.

DNA staining

“Different diseases can affect the cell cycle differently—you can speed up the process, you can see an accumulation in a different phase of the cell cycle. And different drugs will have different effects on the cell cycle as well,” says Timothy Bushnell, scientific director of the University of Rochester Medical Center Flow Lab. “Understanding how a cell goes through a cycle and how it can be perturbed, or where it may be accumulating, is important.”

Perhaps the most common way to query the cell cycle by flow cytometry is to label the DNA with a fluorochrome such as propidium iodide (PI). The principle is simple: diploid cells in G1 phase will have 2N chromosomes—in other words, half the amount of cells in G2 or M phase, which have a 4N chromosomal complement. Cells in S phase, which are in the process of synthesizing new chromosomes (new DNA) have an intermediate amount. Since PI will label the cells in proportion to their DNA content, the percentage of cells in each phase can be read off a histogram.

Flow cytometry generates unprecedented statistical power to monitor the cell cycle of large numbers of cells in a high-throughput manner.

Bound PI will fluoresce green upon excitation with the blue (488 nm) laser that nearly every flow cytometer is equipped with. Cells need to be permeabilized prior to staining with PI, and because PI stains all double-stranded nucleic acids, it’s important to treat the cells with RNAse to get an accurate measure of DNA content.

Yet sometimes a two-color experiment—looking at the expression of green fluorescent protein, for example—is warranted. Or a protocol that doesn’t involve fixation, or permeabilization, or RNAse, is desired. Or cells need to be kept alive to be sorted and re-cultured for downstream functional assays.

A host of other fluorescent dyes have become available—ranging from inexpensive, ubiquitous chemicals such as DNA-specific 4′,6-diamidino-2-phenylindole (DAPI) and Hoechst stains to proprietary dyes such as Thermo Fischer Scientific’s Vybrant® Dye Cycle™ Stains and BioStatus’ Draq series—with combinations of properties that may prove useful in different contexts.

Hoechst 33342, for example, is a cell-permeable dye that seeks out adenine/thymine-rich regions of double-stranded DNA, and is optimally excited by a UV laser. It can be used to stain live cells, and in fact is commonly used in the dairy industry to sort X-bearing sperm, points out Derek Davies, head of the Flow Cytometry Facility at The Francis Crick Institute. Live cell dye cycle stains are available in a variety of colors, “so you’re not stuck with using a laser that you’d really like to use for something else,” notes Helen Fleisig, Thermo Fisher Scientific’s flow cytometry product manager.

But are they cycling?

Simply staining cells for DNA content does not allow for discrimination between G1 and early S phases, nor between late S and G2/M phases. Cells actively synthesizing DNA will incorporate nucleoside analogs such as bromodeoxyuridine (BrdU), indicating that they are cycling. “By altering the way that you add it to the cells you can also use it to assess the duration of cell cycle phases,” says Davies.

BrdU detection requires intracellular staining with a fluorescent antibody—kits are available—which in turn requires a harsh procedure to unwind the DNA to make it accessible. As an alternative, several companies now offer the option of allowing the cells to incorporate 5-ethynyl-2´-deoxyuridine (EdU), which can be detected using click chemistry. The benefits, according to Davies, are that it is faster, does not require an expensive antibody, and that the treatment is much gentler on the cells. “It’s more compatible with other things.” And while “you can do it yourself,” he finds it’s much easier to use a kit—available from multiple vendors— “where everything is QC’d.”

Thermo Fisher Scientific also offers Click-iT® Plus EdU, where the “Plus” indicates “it will work with protein dyes such as GFP and RFP, as well as more sensitive tandem dyes,” says Fleisig. “The regular Click-iT EdU does not work particularly well with those other protein dyes if you’re multiplexing in a panel.”

To examine not just whether, but how many times in a given period, a cell has proliferated—often in conjunction with a DNA dye—researchers will typically use a dye such as carboxyfluorescein succinimidyl ester (CFSE) to stain intracellular amines. The dye is passed along to daughter cells, with the level of fluorescence halving each generation. Newer derivatives are available in multiple colors. Bio-Rad’s CytoTrack™, for example, “is actually an improvement in that it can resolve ten generations (instead of eight), and there is a more consistent generation drop than CFSE,” explains Brenda Karim, product manager at Bio-Rad.

Tell me more

DNA content analysis—perhaps with a proliferation dye and possibly a viability stain and maybe some surface immunophenotyping to distinguish among heterogenous populations—provides sufficient information for most diagnostic or drug discovery purposes. While fixation and permeabilization to look at intracellular proteins requires several additional hours, says Bushnell, “in a lot of cases you’re looking at perturbations of the cell cycle, which can come out of a simple Hoechst-type dye.”

Yet depending on the questions being asked, researchers may want to delve further into the minutiae of the cell cycle. Quiescent cells (such as stem cells) in G0 have identical DNA content as G1 cells, for example, but the two can be distinguished by the relatively greater RNA content in active cells (as can be queried by staining with Pyronin Y), Bushnell points out.

Similarly, while DNA content and proliferative status cannot distinguish G2 from M phases, staining the cells for phosphorylated serine 10 of histone H3 “appears to be the best marker for mitosis.”

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Cyclins and cyclin-dependent kinases (Cdks) can be used to mark checkpoints. “A good way of telling whether your cell cycle has become dysregulated” might be to look for the unscheduled expression of cyclins or Cdks “at particular phases that you don’t expect,” remarks Davies.

While many of these same analyses (and then some) can be done by microscopy on single cells, flow cytometry generates unprecedented statistical power to monitor the cell cycle of large numbers of cells in a high-throughput manner. Whether querying DNA content alone or simultaneously examining a battery of parameters, the answer is likely in the flow.

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