Flow Cytometry and Cell Sorting Comparison

Since their inception, flow cytometry and flow cytometric cell sorting have become central technologies in the fields of cell biology and immunology, revolutionizing our handling of cell populations, and leading to significant research and clinical advancements. But while both techniques use the same basic principle to differentiate cells based on their optical properties, there are some fundamental differences between them which result in distinct functionality.

High-throughput analysis

”The core components, fluidics, optics and electronics, of every flow cytometer are the same, whether the instrument only performs cell analysis or is capable of purifying different populations through cell sorting,” explains Monica DeLay, Director of Field Applications at Cytek.

In flow cytometry, heterogeneous populations of cells are analyzed based on their physical and chemical properties. Cells are classified into groups, and the proportion of each cell type is measured. The fast, efficient, and cost-effective nature of flow cytometry, in addition to the fact that multiple cell parameters can be measured simultaneously, means the technique is ideal for the high-throughput characterization of large cell populations, and has been used in hundreds of research areas.

”By far, the most common application of flow cytometry analysis is immunophenotyping,” describes DeLay. Allowing quick and easy phenotyping, the technique is an invaluable tool in the diagnosis and prognosis of many types of leukemias and lymphomas. Numerous aspects of cell biology can also be easily studied with flow cytometry; other common uses include cell proliferation assays, cell cycle analysis, apoptosis detection, and the measurement of intracellular calcium flux.

Once generated, however, “data serves as the end point for an analyzer,” Monica DeLay explains. After cells have passed through a flow cytometer they are discarded without cell recovery.

Cell sorting is just the beginning

In flow cytometric cell sorting the process goes further. “At a fundamental level, a cell sorter is a flow cytometer that can separate cells,” explains Richard Cuthbert, Flow Cytometry Product Manager, Bio-Rad Laboratories. “Every sorter is in fact a cell analyzer but not every analyzer is a sorter.”

While in flow cytometry, cells are measured and discarded after data collection, in “sorting, cells can be separated according to what is seen in the analysis,” Cuthbert highlights. After cell sorters separate cells based on their fluorescence properties, a subset of cells is retained for further analysis. “This opens up a huge range of additional applications that can be performed post sorting,” continues Cuthbert.

One such application is in the advancement of immunotherapy. Lymphocyte and stem cell populations isolated by cell sorting now routinely play a pivotal role in cancer research and drug discovery and development. Other downstream applications of sorted cell populations include genetic screening and prenatal diagnosis, establishing cells lines for further biological research, in vitro drug testing, and proteomics. “The power of cell sorting is enhanced by many complementary methods that utilize both bulk purified populations and single sorted cells,” explains DeLay. “In fact, parallel development of single-cell omic technologies has expanded the use of cell sorting to other fields including developmental biology and neurobiology.”

With the increased functionality of cell sorting, however, comes some limitations. “Cell analyzers tend to be easier to use, easier to set-up, and easier to maintain,” explains Cuthbert. Cell sorting, on the other hand, requires more expensive, specialized equipment and training to get good results.

As cell sorting typically isolates cells through positive selection, a larger starting cell population is required to ensure a sufficient number of desired cells. In addition, the process of gating or setting the fluorescent thresholds to separate cells can take time to optimize, further increasing the risk of losing cells. Then there is the process of separation itself, which can impact cell viability. Cell death can slow sorting, lead to inaccuracies, and impact the effectiveness of downstream applications. And with clinical applications demanding high levels of cell purity, stringent quality control must be used to ensure cells meet requirements.

But despite their complications, the critical advantage of separating cells means cell sorting is an essential part of many experimental workflows, DeLay summarizes, emphasizing that “isolated cells can be used in a variety of in vivo and in vitro assays” not previously possible.

Together we are stronger

While flow cytometry offers fast, cost-effective characterization of large cell populations, and cell sorting provides essential samples for downstream applications, deciding which technology is best for you ultimately depends on your research needs. But does there need to be a choice? Cell sorting and flow cytometry can be used together to help gather further biological insights.

But what for the future? “Flow cytometry is a technology that is constantly developing, both in terms of instrumentation and reagents,” explains Cuthbert. “With the emergence of full-spectrum flow cytometry, we can now distinguish broad cell populations based solely on their autofluorescence signatures,” Monica DeLay continues, allowing samples from complex tissues or marine environments to be studied. And with new technologies such as label-free isolation and spectral cytometry continuing to evolve, the use of flow cytometry and cell sorting will remain an important component of cell analysis.

Table. Flow cytometry vs Cell sorting