Although flow cytometry first appeared (as fluorescence-assisted cell sorting) in the late 1970s, the last three years have seen a surge of innovation in instrumentation and applications. A primary factor in this renaissance has been increasing demand for immunophenotyping cells undergoing development, which involves characterizing cells by virtue of their surface antigens. Immunophenotyping sheds light on how the body responds to diseases and drug regimens, and is applicable to both mature and stem cells.
Factors driving flow cytometry as an instrumental technique include innovations in laser and detector technologies, “fail-safe” modes that make cytometric data more robust (e.g., between multiple instruments and multiple labs) and greater ease of use, portability, and affordability. Flow cytometry has become a method of choice for preparing cells for analysis, characterization, and even culturing.
In particular, laser and detector technologies make possible a broad, rich variety of reagents for detecting specific cell types. “And this has resulted in a concomitant increase in the experimental complexity and the richness of experimental data,” notes J. Clark Mason, Ph.D., senior director of global marketing, BD Biosciences (San Jose, CA).
Moving in two directions
Yet the intricacy of experiments has been largely offset and even enabled by the evolution of instrumentation, workflows, and data analysis—at least at some level—toward greater accessibility. These benefits have had a stabilizing effect on flow cytometry in general, to the point where results achieved at different locations by different groups are now comparable.
Flow cytometers that are relatively easy to use, portable, and affordable have caused an upsurge of interest in nontraditional markets such as marine biology, the study of microorganisms and plants, and environmental analysis. Flow cytometers are now more accessible than ever, both inside and outside core labs.
“Developments in genomics and proteomics focusing on single-cell analysis at the single-copy level are another driving force for growth,” says Dr. Mason. Next-generation sequencing and sequence detection with real-time PCR has caused a “new appraisal” of flow cytometry’s potential to sort single cells—particularly those existing in very low abundance.
T. Vincent Shankey, Ph.D., principal staff advanced research scientist at Beckman Coulter (Miami, FL), agrees to a point, but sees flow cytometry as “running in two directions, simultaneously toward simpler and more complicated instrumentation.” Major manufacturers are indeed creating “everyman”-type products that are less demanding and targeted toward both expert and occasional users. “But everyone is also interested in instruments that analyze more and more independent fluorophors, which is the opposite trend.”
With apologies to basic users everywhere, it is the complex end of the application spectrum that excites Dr. Shankey. He contrasts flow cytometry with NMR, which he says “performs a certain interrogation, from which you get a limited set of answers. If you don’t understand the data, you can find someone to help.”
Flow cytometry, he says, requires a very broad range of skills that include knowledge of antibodies-antigen interactions, dye chemistry conjugates, electronics, and signal processing, not to mention some information about cell biology and data analysis and interpretation.
Some flow experiments are indeed straightforward; for example, how many cells are alive or dead. More complex is the question of how many cell types may be identified (and perhaps separated) using a particular set of antibodies to specific surface antigens. “The problem is that if half the cells are dead and you don’t know it, your results will be different. With NMR there aren’t as many ‘maybes.’”
Dr. Shankey notes the trend in dumbing down instrumentation and methods. “I’m all for that. Flow cytometry should not be a sacred shrine into which only a select group of scientists may enter.” However, that is not where the cutting edge exists today.
Most users, in fact even most core facilities, run routine experiments 80 percent of the time. “There is no point taking out the Ferrari when you can drive it only 20 miles per hour.” However, the immunology literature is now dominated by extremely complex experiments based on flow cytometry, using multiple colors, multiple gatings, and examining multiple low-population cells responding to different stimuli over time. “Those require the Ferraris, the more sophisticated instruments.”
High-end workflows increasingly rely on automated sample preparation and, at the back end, more automated data analysis, although Dr. Shankey describes data handling for complex experiments as a “logjam” that is in “desperate need of fixing.” Current cell analysis, even at the high end, is based on applying multiple conditions or “gates” on which cells are counted. “And that’s one of the areas where the field is stuck. We’re only viewing data in two dimensions instead of in high-dimensional mathematical constructs.”
Digging deeper inside cells
While flow cytometry has become the go-to tool for immunology labs, the explosion in reagent R&D has made other types of investigations possible. “Flow cytometry was once largely used to analyze cell surface receptors, but now more functional assay types are possible,” says Mike Olszowy, Ph.D., director of flow cytometry systems at Life Technologies (Eugene, OR). Some of these include assays for cell proliferation, calcium flux, cell cycle analysis, and events occurring deep inside cells.
Life Technologies’ acquisition of Molecular Probes in 2003 and Dynal in 2005 consolidated its leadership in nontraditional fluorescent probes for flow (and other) cell-based investigations. Cell- Trace™ Violet, used for studying cell proliferation, is one such reagent.
Life has also introduced an instrument, the Attune®, that speeds up cell analysis by a factor of ten. Attune uses a new celldirecting technique known as “acoustic focusing,” which causes cells to line up narrowly as they pass through the flow cell. Normally, cells passing through in high-flow mode spread out within the flow stream. Some hit the “sweet spot” of the laser detector, but many do not, causing considerable variability. By squeezing cells into a very narrow path, acoustic focusing forces the cells into the narrow window of optimal laser operation.
For additional resources on flow cytometry, including useful articles and a list of manufacturers, visit www.labwrench.com/flowcytometers