Over the past few decades, technologies like flow cytometry and fluorescence-activated cell sorting (FACS) have revolutionized our understanding and manipulation of cell populations. Since their inception, there have been significant advancements and applications, from clinical diagnosis to biomedical research, propelling a new era of cellular analysis and handling.
The terms flow cytometry and FACS are often used interchangeably. However, this is not accurate. Namely, FACS is a subtype of flow cytometry that allows cells to be sorted and retained rather than simply counted, analyzed, and disposed of. While all cytometry harnesses light scattering techniques as cell samples flow through a beam of light, there are differences that distinguish FACS from other types of cytometry.
Flow cytometry’s primary objective is to gather a comprehensive understanding of the various cell populations within a particular sample. In this way, flow cytometry lets researchers gather statistical data about individual cell populations from a heterogeneous mixture.
A flow cytometer funnels sample cells individually through a narrow channel within a fluidics system. The cells then pass through the optics system—a series of lights and sensors—that can characterize cells based on the way light is reflected and refracted as cells pass through it. These changes in the light trajectory are commonly referred to as vertical and horizontal light scattering and are measured by the sensors within the system. All of this data is stored and processed by the flow cytometer’s computational system before the cells are processed. The output provides a statistical representation of populations within the cell sample.
Target populations can also be identified with more specificity by using the relationship of antibodies and antigens to create a system of tags. By fusing antibodies with fluorescent dyes and allowing them to bind to antigen-presenting cells, the cytometer can recognize tagged cells as distinct from the rest of the sample.
Different fluorescent dye compounds can be used to distinguish specific cellular structures or cell populations. By differentiating the light wavelengths emitted from the fluorescent markers, the flow cytometer can quantify several characteristics of each individual cell simultaneously. Moreover, light scattering—both forward and side-scatter—provides intricate details about a cell’s size, granularity, and internal complexity.
Flow cytometry is used for indexing cell samples, including immune-rich biological samples like bone marrow, peripheral blood, and other biofluids. These types of samples often contain a high concentration of immune cells, which are valuable for research and diagnostic assays to advance scientific discoveries, gather information about a particular patient, or help determine the efficacy of new therapeutics and treatments.
Flow cytometry has also been used in several groundbreaking research areas, such as studying the dynamics of HIV infection within T cell populations or understanding the heterogeneity within tumor cells. Using flow cytometry data, these studies provide insights into potential therapeutic strategies.
Fluorescence-activated cell sorting (FACS) is a subset of flow cytometry with the end goal of counting, sorting, and saving a subset population of cells from the initial sample. FACS techniques read fluorescent-bound antibody probes and physically partition the heterogeneous cell sample into separate populations. Isolated cells can then be used for further research or culturing.
FACS protocols are similar to general flow cytometry using fluorescent antibody tags. However, while the cells are funneled one by one through the flow cytometer, FACS also sorts the cells in real-time based on the intensity of excited light of their fluorescent markers.
Instead of being discarded after data collection, an extra modification separates single cells into containers of specific cell types using targeted electrical currents that alter the fluid flow pattern. These isolated cell samples can be studied and used for later experimentation. Some examples of these downstream applications include genetic analysis, in vitro drug testing, and initiating cell lines for further biological research.
However, FACS is not particularly gentle on cells. The sorting process may impact cell viability and function, which can limit the effectiveness of these applications. Additionally, applications for clinical have stringent requirements for cell purity. The possibility of sample cross-contamination requires rigorous quality control during the FACS process to ensure the utility of the sorted cells later on.
FACS is primarily used when the goal is to separate cells into isolated populations for later use—as compared to general flow cytometry, where data is preserved, but the cell samples are discarded. Cell sorter applications include isolating stem cells or lymphocyte populations from white blood cells. Specifically, it has been integral in research developing CAR-T cell therapies for cancer treatment. By isolating T cells that are then genetically modified to target cancer cells, FACS has played a pivotal role in advancing immunotherapy, demonstrating its vital utility in the field of oncology research and treatment development.
FACS is a popular method of cell separation because it can sort large, diverse samples. When dealing with relatively complex mixtures, FACS flow cytometry allows researchers to effectively sort through the sample and retain just the cells of interest. However, depending on the sample and how it was prepared, FACS can take significant time to process and sort the sample, and throughput is limited. It also requires access to expensive equipment and the trained personnel to use it.
FACS often requires a large initial cell population because it usually isolates through positive selection, only targeting the desired cells and leaving the rest to flow through to disposal. If cells are in clusters or cannot be clearly identified with the fluorescent tags, they are discarded as false negatives.
In addition, there is subjectivity in setting the fluorescent thresholds that separate the cells, a process called gating. The retention relies on both the quality of the fluorescent probe and skill of the user, creating a risk of losing many cells throughout the process if not properly optimized. To compensate, researchers usually start with a high number of cells with hopes of ending with a sufficient population.
The process can also be harsh on sensitive cells, reducing overall throughput as the fast-moving fluids rupture cell membranes. This is called cell shearing. As more cells die, their extracellular debris begins to pile up in the tube and cause blockages. These blockages can lead to inaccurate sorting and even more cell death. It also slows the processing of cells and can bind to future cells, causing additional false positive readings.
Depending on the sample type, it may be beneficial to use a different cell separation method or to optimize your sample prior to flow cytometry. Removing dead cells and enriching for cells of interest before beginning the flow sort can help improve the input sample quality and resulting separation. One of those ways is to pair them with BACS™.
Buoyancy-activated cell sorting (BACS™), Akadeum’s patented approach to cell isolation, offers a novel way to isolate the target of interest using science so simple it floats. Akadeum’s microbubbles are small, solid particles that float. They can be functionalized to bind to any number of targets of interest, including cells, proteins, nucleic acids, and more. The microbubbles are simply mixed into the sample, where they engage and bind with their targets, then isolate those targets using gentle, floatation-based separation.
Thanks to their inherent buoyancy, microbubbles simply float to the top of the container, bringing their bound targets with them. This can be done to positively enrich target cells or deplete contaminating cells by selecting negative populations and leaving cells of interest untouched.
Akadeum has taken technology that traditionally requires additional equipment and consumables and put it into a single container for separation—no magnets, columns, or limitations. Microbubbles offer increased throughput, higher sensitivity, and greater recovery, all while eliminating the need for expensive equipment. Critically, the microbubble workflow is exceptionally gentle. This is especially important when enriching for delicate target cells and cells of low abundance.
For flow cytometry and cell sorting, BACS™ can be a critically helpful sample preparation step to reliably achieve a greater population of healthy, viable cells for downstream use. Using BACS™ to remove contaminating cells and enrich for cells of interest before running the sample through a flow cytometer can enhance efficiency, reduce cell death, and improve analytical results.
Depending on the sample composition and target cell population, it may be beneficial for researchers to use BACS™ instead of FACS. Especially with small, rare cell populations, BACS™ enables higher retention efficiencies and is incredibly gentle with fragile cells. Because microbubble enrichment occurs directly in the sample container, a BACS™ workflow eliminates issues like shearing, gating, or blockages.
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