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.
Researchers measure cellular proliferation for any of a number of reasons. They may wish to determine the extent of antigenic T cell stimulation, for example, or quantify how well a compound inhibits tumor growth. Even for routine cell-culture QC, like plotting growth under different conditions or just knowing when to passage a culture, it’s a good idea to quantify cellular reproduction.
There are many ways to measure proliferation, some more direct than others. Some measure DNA replication or protein synthesis, and others look at metabolic activity or simply the percentage of the culture-dish plastic that’s covered. These assays exploit a range of lab tools, from antibodies to counters to microscopes. Here we examine how imaging technologies let researchers gather far more information about what their cells are doing, and which cells are doing it.
Imag(in)e that!
Imaging modalities themselves encompass a range of modalities and instrumentation, perhaps the simplest of which is a bright-field microscope with a camera to document what is happening in the field of view. With such a setup, it’s possible to determine how many cells occupy a plate or a well, or a plate’s or well’s percent confluence, in a label-free manner and without interfering with the culture. Combine this with software that can distinguish cells from background, move the camera (or sample) and perform basic calculations, and you have an automated proliferation assay.
Molecular Devices’ SpectraMax® MiniMax 300, a user-installable imaging cytometer add-on to the company’s SpectraMax i3 Multi-Mode microplate reader, has such capabilities. The MiniMax 300 comes with pre-defined settings, enabling it to recognize many cell types from the transmitted light images. It can also be taught to recognize other cell types using supervised machine learning, says Molecular Devices application scientist Cathy Olsen. The MiniMax module also boasts green and red fluorescence channels , allowing it to simultaneously track the nucleus with FITC and mitochondrial activity with Cy5, for example, or to distinguish between cell types.
Dedicated imaging instruments are on the market, as well. Some, such as Thermo Fisher Scientific’s Tali® Image Cytometer, are designed for single samples of suspension cells, pipetted onto proprietary slides. Others, such as Nexcelom’s Celigo® S Imaging Cytometer, can handle adherent or suspension cells in a variety of formats, including T-flasks and 1,536-well plates.
Beyond counting cells and calculating confluence, such instruments support a host of fluorescence-based assays; these include viability, apoptosis and cell-cycle analyses.
More than just numbers
Many common assays that tell whether cells are healthy, growing and reproducing have fluorescence versions, enabling users to query individual cells (rather than populations or lysates) via imaging. Metabolic activity, for example, can be assayed using fluorogenic mitochondrial pH indicators; cell-impermeable dyes such as propidium iodide can measure membrane integrity; and DNA replication can be assessed by quantifying incorporation of the nucleoside analogues BrdU (using fluorescent antibodies) or EdU (via Click chemistry). Cell-cycle markers can be tagged with fluorescent antibodies, as well.
But are these measuring proliferation?
Randy Wetzel, director of cytometry at Cell Signaling Technology, says many assays only measure surrogates for proliferation. Cells can continue to metabolize, and to increase or change in size, without ever experiencing mitosis. They can enter into mitosis and arrest at some stage of the cell cycle. Using nucleoside analogues (including radioactive thymidine) or membrane or protein dyes that get split between daughter cells gives a historical measure of proliferation rather than identifying which cells are currently proliferating. Even cell counting is more a measure of the balance between proliferation and cell death than it is of proliferation per se, he notes.
The closest would be an antibody against something like phosphorylated histone H3, an activated protein that is “only on when they’ve made it into the M phase and they’re actually dividing,” he explains. “You can look at the dish and say, right now there are 4.5% of the cells in mitosis.”
HCA
Among the selling points of imaging is the ability to multiplex. With cancer studies, for example, it’s important to know more than whether a drug prevents proliferation. You want to “look at a variety of markers all at the same time and then really do correlations to find out if A is happening before B is happening before C, or whether something else is affecting what’s going on in the cell,” says Scott Keefer, product marketing manager of cellular imaging and analysis product lines at Thermo Fisher Scientific.
And when it comes to that kind of analysis, high-content analysis (HCA) “is kind of at the top of the food chain” in terms of sophistication, Keefer says.
An imaging cytometer like the MiniMax sports a single 4x objective and looks at samples cell by cell. In contrast, it’s not unusual for an HCA platform to offer multiple objectives from 4x up to 100x, as well as other optical enhancements, to “look more at the subcellular level,” says Olsen. And unlike traditional flow cytometry, HCA “can measure very sophisticated things like morphologies, like translocations—very subtle phenotypes,” Keefer says.
Live cells
Sometimes it’s important to determine whether cells are proliferating over time—perhaps in response to treatment—rather than simply taking an end-point measurement. But repeatedly transporting plates between a microscope and incubator can negatively impact the assay, so many HCA instruments provide control of environmental conditions such as temperature, humidity and CO2, allowing live-cell assays to be followed over time.
Another option—one that doesn’t tie up an expensive HCA—is the IncuCyte ZOOM from Essen BioScience. This live-cell imager sits inside an incubator and can simultaneously accommodate and remotely monitor up to six microplates, or a variety of flasks or dishes, using high-definition phase contrast and two fluorescence channels. The company also has developed a line of lentivirus-based reagents to transiently or stably label cells with fluorescent proteins “in a non-perturbing way, so that it doesn’t affect their proliferation,” explains Essen BioScience R&D project manager Dan Appledorn.
Bottom line: You want to know if your cells are proliferating, and at what rate. With imaging-based approaches, you have the tools to figure that out.