May 2021 Share
As medical research becomes more advanced, scientists must use more complex technology to identify, separate, and prepare cells for downstream applications. Two popular methods for this include flow cytometry and fluorescence activated cell sorting.
In the field of cell sorting and analysis, the terms flow cytometry and fluorescence activated cell sorting (FACS) are often used interchangeably. While each harness light scattering techniques and flowing cell samples through a laser beam, there are some key differences that distinguish the two processes.
The primary objective of flow cytometry is to gather a comprehensive analysis of a particular cell sample. Flow cytometry allows researchers to gather statistical data about individual cell populations from a heterogenous mixture.
Flow cytometry analyzes cells using a device called a flow cytometer. A flow cytometer funnels sample cells through a narrow channel one by one with its fluidics system. The cells then pass through the optics system – a series of lights and sensors which can detect cell characteristics based on vertical and horizontal light scattering. All of this data is stored and processed by the third and final component of a cytometer, the electronics system, before the cells are disposed. These three parts work together to provide a statistical representation of populations within the cell sample.
Target populations can also be identified with more specificity by using antibodies and antigens. By tagging antibodies with fluorescent dyes and binding them to antigen-presenting cells, the cytometer is able to recognize them as distinct from the rest of the sample.
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 immunology research and diagnostic analysis to advance scientific discoveries, gather information about a particular patient, or help determine efficacy of new therapeutics and treatments.
Fluorescence activated cell sorting (FACS) analysis is a derivative of flow cytometry that proceeds in a slightly different direction. The primary objective of FACS flow cytometry is to physically sort a heterogeneous cell sample into separate populations. Isolated cells can then be used for further research.
The process of FACS is similar to flow cytometry but involves an extra step. While cells are still marked with fluorescent antibodies and funneled one by one through a flow cytometer, they are also sorted. Instead of being discarded after data collection, an extra device modification separates unique populations into their own containers. These isolated cell samples can be studied or harnessed for practical applications.
This 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 being processed and how it was prepared, FACS can take a significant amount of 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.
The FACS procedure also requires a large initial population because it isolates through positive selection, targeting the desired cells. Due to the potential of losing many cells throughout the process, researchers must start with many cells if they hope to end with a sufficient population. Overall throughput is also reduced by shearing.
Shearing occurs when cell membranes rupture due to the fast-moving fluids. 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. Depending on the cell population, it may be beneficial to use a different cell separation method or to optimize your sample prior to FACS processing to remove dead cells and enrich for cells of interest before beginning the flow sort.
FACS is a cell separation technique, meaning it’s used when the goal is to separate cells into isolated populations. The reason FACS is used as opposed to flow cytometry is because the researcher has downstream use for the sorted cells. Flow cytometry helps to gather data; FACS prepares samples for downstream applications. Those applications could include further research or the isolated samples being used for medical treatment.
While both flow cytometry and fluorescence activated cell sorting can prove valuable by themselves in a laboratory setting, there are ways to improve their overall benefits. One of those ways is to pair them with BACS.
Buoyancy-activated cell sorting (BACS), Akadeum’s patented approach to sample preparation, 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 are 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 with their targets, then isolate those targets using gentle, floatation-based separation. The microbubbles, using their inherent buoyancy, will simply float to the top of the container, bringing with them their bound targets.
Akadeum has taken technology that traditionally requires additional equipment and consumables and put it into a single container for self-separation. No magnets, no columns, no limitations. Microbubbles offer increased throughput, higher sensitivity and greater recovery, and eliminate 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 critical step in sample preparation that helps to achieve more reliable results and 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.
When it comes to FACS specifically, BACS can be used to enrich the sample prior to cell sorting, after cells are sorted to further isolate them from debris, or in some cases, completely replace FACS cytometry as a separation method. 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 throughput and is incredibly gentle when dealing with fragile cells. Because microbubble enrichment occurs directly in the sample container, a BACS workflow eliminates issues like shearing or blockages.
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