Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
To an immunologist, cell proliferation is often the principle readout of T-cell stimulation. Stimulated T cells create clonal duplicates of themselves that are able to respond to the same stimulus, creating an army of cells on the lookout for their specific antigen.
There are myriad flow cytometric methods researchers can use to see if cells are or have been replicating, and these generally fall into two different camps. Some assays, relying on reagents like nucleotide analogs (e.g., bromodeoxyuridine, BrdU), DNA dyes (Hoechst and propidium iodide, PI) and antibodies to cell cycle-regulated proteins (e.g., Ki-67), are typically used to discriminate quiescent from dividing cells, and sometimes to identify cell-cycle stage. Given the length and timing of the labeling pulse, or in combination with one another, these methods can also be used to quantify cell turnover.
The second camp, sometimes termed “generational analysis” assays, indicates more directly how many times a given cell has divided since it was labeled. Here, cells are labeled with a vital dye that permanently attaches to the cell. When the cell divides the dye divides with it, and the signal intensity halves each round to indicate how many divisions have occurred—but only out to six or eight generations; after that, the signal typically is too dim to be distinguished from background autofluorescence.
Here we discuss the power and pitfalls of such assays.
Go with the flow
In flow cytometry fluorescently labeled cells pass single-file through a flow cytometer, which excites the fluorophores and quantifies the fluorescence emitted at a given wavelength. The fluorophores can be encoded into the cells’ DNA (e.g., using green fluorescent protein, GFP), conjugated to an antibody or otherwise react with particular cellular components.
Not only does flow cytometry allow cells to be queried individually, “it’s a very powerful technique that allows us to measure many different characteristics at once,” says Robert Balderas, vice president of biological sciences for flow cytometry instrumentation developer, BD Biosciences. “As a platform it allows one to look at size and granularity of a cell, what’s on the cell, what’s in the cell or what’s made by the cell.”
Most importantly, Balderas says, flow cytometry “gives us a chance to find a needle in the haystack." Researchers can use the technique to find unique cell types within a heterogeneous population, for instance by staining for differentiation markers, cell-surface activation markers, intracellular proteins or secreted cytokines.
Such immunophenotyping not only can differentiate between Th1, Th2 and T-regulatory T cells, but can also determine how markers on each subset vary, for example after three versus six post-stimulus divisions.
Proliferation dyes
The most common ways to track proliferation in T cells are to attach fluorophores to intracellular proteins (“protein dyes”) or allow them to intercalate into the cell membrane (“membrane dyes”), says Paul Wallace, professor of oncology and director of the department of flow and image cytometry at the Roswell Park Cancer Institute in Buffalo, NY.
Protein dyes are typified by vital dyes such as carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), an uncharged species which can easily transit the cell membrane. Once inside the cell CFDA-SE is cleaved by endogenous esterases, resulting in a reactive aldehyde that indiscriminately binds to amines found on proteins. When the label is excited by a blue laser it fluoresces green. But other, similar, protein dyes have been developed more recently with a host of different spectral properties, allowing them to be used in conjunction with fluorescent reagents with which CFDA-SE may interfere.
CFDA-SE staining is a relatively straightforward procedure, and the cells will yield beautiful histograms with peaks—corresponding to cells having undergone a given number of divisions—punctuated by valleys. And because the compound only interacts with a small subset of proteins, it appears to have little effect on cellular function, notes Steven Porcelli, professor of microbiology and immunology and scientific director of the flow cytometry core facility at Albert Einstein College of Medicine. But there can be some effect, he notes, "and that imposes a limit to how fluorescent you can make the cells.” Porcelli therefore recommends that researchers optimize the assay by titrating CFDA-SE concentration.
Membrane dyes are typified by Sigma-Aldrich’s PKH series, but versions with different spectral properties are also available from various manufacturers. A long hydrocarbon tail attached to the polar fluorescent head group allows the membrane dyes to rapidly intercalate in the lipid bilayer and to be stably but non-covalently retained in the cell.
Membrane dyes seem to have no impact on cellular function and thus allow very bright cells to be achieved (up to the limit of compromised membrane integrity). They are “so lipophilic that they just want to go right into the membrane,” says Wallace, and they can be used almost immediately for kinetic studies (unlike protein dyes, which take up to a day to stabilize). Yet this also means the process is trickier, Wallace says, and if done improperly can lead to cells that are not homogenously stained. "It’s a little bit more finicky and takes a little more practice to get good at.”
In vivo vs. in vitro
It’s easy to see how cells can be stained in vitro, and then stimulated and cultured in vitro or adoptively transferred into an animal host for in vivo studies. But not all studies are amenable to cells being taken out of an animal, labeled and reintroduced into live animals, notes Evan Jellison, director of flow cytometry and assistant professor of immunology at the University of Connecticut Health Center.
In such instances cells must be labeled in vivo. Researchers will typically pulse the animals with BrdU (by injection or simply putting it in the drinking water) to ascertain which cells have replicated their DNA during the pulse period, he says. Yet to visualize BrdU by flow cytometry it’s necessary to first permeabalize the cell and nuclear membranes and then denature and query the DNA with a fluorescently labeled anti-BrdU antibody.
An alternative is Life Technologies’ new Click-iT® Plus EdU Alexa Fluor® 647 Flow Cytometry Assay Kit. Here the animals are injected with the thymidine analogue EdU (ethynyldeoxyuridine), which contains one component of the Click reaction. After harvesting the cells, EdU is labeled with an azide-derivatized fluorophore (the second component). This detection reagent is small enough—about 100 times smaller than a typical antibody—to find its target without having to first denature the DNA, making the protocol far faster and simpler, says Mike Olszowy, R&D leader for biological flow cytometry at Thermo Fisher Scientific, Life Technologies’ parent company.
And there are other ways to determine whether T cells have reacted to a stimulus, too—counting cells with a hemocytometer and measuring the uptake of tritiated deoxythymidine are tried-and-true in vitro methods, for example. Yet multi-parameter flow cytometry affords the opportunity not only to determine whether cells have been stimulated to proliferate, but which cells have proliferated, by how much they have done so and what changes they have undergone in the process. A banquet of fluorescent reagents allows the proliferation assay to be both powerful and nuanced indeed.