Fluorescence Activated Cell Sorting (FACS)

Fluorescence activated cell sorting (FACS) is a specialized method of flow cytometry that uses fluorescent markers to target and isolate cell groups based on cell surface markers. This method works in the fact that antigenic ligands, like proteins and carbohydrates, give each cell a unique surface phenotype, which can be used as specific targets.

FACS provides researchers the ability to separate cells based on physical characteristics like size, granularity, and cytokine expression. This cell sorting technique has highly dynamic capabilities, may allow for high throughput testing, and is commonly used in hematopoiesis, oncology, and stem cell biology research. FACS is a flow cytometric procedure that provides the ability to also measure and characterize multiple cell generations within a sample. In this way FACS can simultaneously gather expression data and sort cell samples by the required input variables.

 

Differences between Flow Cytometry & FACS

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Both flow cytometry and FACS techniques use fluorescence and other properties to highlight the differences in cell surface or intracellular components of the different cell types in a mixture. Both techniques acquire fluorescent, forward-scatter, and side-scatter data. Flow cytometry and FACS can both be used in a wide range of applications in medicine, immunology, molecular biology, pathology, and genetics.

However, there are several differences between them:

Basis of differentiation	Flow Cytometry	FACS
Definition	Flow cytometry is an analytical cell biology technique used to identify and study the characteristics of cells in a heterogeneous mixture. It uses differential light scattering properties unique to each cell type in the mixture to determine the number and size of cells and nucleic acid content of the cells.	FACS (fluorescence-activated cell sorting) is a specialized type of flow cytometry that facilitates the sorting out cells in a heterogeneous mixture into two or more types. It uses fluorescent-labeled antibodies to specifically identify components of different cell types
Type of technique	This is an analytical cell biology technique.	This is a specialized type of flow cytometry.
Sampling method used	The process uses the differential light-scattering properties of cells to collect the necessary data.	The process uses highly specific antibodies tagged with fluorescent dyes to distinguish between cell types.
Analysis method	A sensor is used to acquire data.	An electromagnet is used to sort the sample.
Sequence	Flow cytometry follows FACS.	FACS is the first step of analyzing a heterogeneous cell mixture. It is followed by flow cytometry.
Function	This technique measures certain cell characteristics such as the number, size, and nucleic acid content of cells.	This technique separates cells from a heterogeneous mixture into appropriate subpopulations.

Applications

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FACS offers a number of applications in the field of research and diagnostics. In immunophenotyping, FACS can identify and quantify multiple populations of cells in a single heterogeneous sample for peripheral blood, bone marrow, and even lymph material. Commonly, FACS is used in a hematological setting in which specific cancer subtypes may be diagnosed. FACS may also be used for the purpose of cell cycle analysis, where cells can be analyzed and measured in all four distinct phases.

Secondary cell-based assays can also be used to further assist with the determination of cell anomalies using certain fluorophores, a common practice throughout single-cell genomics. FACS may additionally be used to assess cell viability and proliferation, commonly performed by labeling resting cells with a membrane fluorescent dye like carboxyfluorescein succinimidyl ester (CFSE). This works by the principle of mitosis; as cells grow and divide, half of the dye is passed to each daughter. By measuring the reduction in fluorescent signal, cellular activation and proliferation can be determined.

The extent of apoptosis and necrosis within a sample can also be identified and distinguished, which may be helpful in determining morphological, biochemical, or molecular changes that occur over time. FACS has also been used to assess membrane potential and interpreting ion flux within a sample. This technique provides the capability of detecting the flux of calcium ions that are drawn into a cell, which thereby measures the activation of associated signal transduction pathways. FACS has a number of other applications, including:

1. Assessing pH changes
2. Evaluating redox state or oxidative metabolism
3. Analyzing chromatin structure and gene expression
4. Gauging total protein expression, lipid population, or enzyme activity
5. Providing light on the extent of DNA replication and/or degradation

Methodology and Principles

 The methodology for a FACS experiment is straightforward.

1. Cells in suspension are prepared with a stain of fluorescently-targeted monoclonal antibodies (mAb) that recognize specific surface markers within the desired cell population.
2. Predetermined fluorescent parameters of the cells of interest can be input into a flow cytometer, and sorting parameters can be adjusted depending on the desired output purity and yield of the sample.
3. The suspension is passed through the instrument as a stream of droplets, each containing a single cell, in front of a laser.
4. The flow cytometer applies a charge to each droplet and an electrostatic deflection system facilitates the assembly of the charged cells into appropriate collection tubes for analysis and quantification.

FACS offers a number of advantages over other methods of cell sorting, and is especially useful for detecting very low levels of protein expression. FACS is the only available purification technique that utilizes size, granularity, and marker detection via fluorescent targeting of intracellular proteins. FACS is largely useful if separation based on differential marker density is required, and the technique provides the ability to negatively select unstained cells, if necessary.

Flow cytometry, in general, requires an adequate number of cells in the starting material, commonly around 1 million, as staining and washing procedures will cause cell loss. It should be stated that the recovery rate of FACS is, on average, between 50-70%, which could pose a disadvantage when working with rare cells. The choice of flow cytometer must also be taken into consideration, as FACS purification requires a strong sorting capacity, with an appropriately coupled software.

Adapted and Integrated FACS Techniques Used in Research

Pairing FACS with other experimental techniques has recently given researchers the ability to better explore human biology. FACS has been used in tandem with targeted analysis of histone modification to better profile primary human leukocytes. This research has helped contribute to knowledge of histone post-translational modification, required for differentiation and maintenance of certain distinct cell types. Some research has also integrated microfluidic devices with FACS to create a miniaturized analytical system that can perform similarly to a flow cytometer. The development of this technology is aimed towards diagnostic applications with the ultimate goal of a low-cost, portable instrument for point of care use.

Product Ordering Information

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References

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Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)
Development of a microfluidic device for fluorescence activated cell sorting
Ultra-high-throughput screening based on cell-surface display and fluorescence-activated cell sorting for the identification of novel biocatalysts
Understanding the Relationship Between FACS and Flow Cytometry
Coupling Fluorescence-Activated Cell Sorting and Targeted Analysis of Histone Modification Profiles in Primary Human Leukocytes
Development of a microfluidic device for fluorescence activated cell sorting
Separation of SSEA-4 and TRA-1-60 Labelled Undifferentiated Human Embryonic Stem Cells from A Heterogeneous Cell Population Using Magnetic-Activated Cell Sorting (MACS) and Fluorescence-Activated Cell Sorting (FACS)