What is the Difference Between Flow Cytometry and FACS

The main difference between flow cytometry and FACS is that flow cytometry allows to rapidly, accurately, and simply collect data related to many parameters from a heterogeneous fluid mixture containing live cells. But, FACS (fluorescence-activated cell sorting) is a derivative of flow cytometry that allows to physically sort a heterogeneous mixture of cells into different populations. Furthermore, flow cytometry uses the differential light-scattering properties of cells to collect data, while FACS uses highly specific antibodies tagged with fluorescent dyes to differentiate between cell types. Moreover, flow cytometry uses a sensor to acquire data, but FACS uses an electromagnet to sort the sample. 

In brief, flow cytometry and FACS are two techniques in analytical cell biology used to profile cells in a heterogeneous mixture. Generally, flow cytometry involves the cell analysis and measuring protein expression as a further analyzing technique of FACS, which involves sorting out of cells in a mixed population. 

Key Areas Covered 

1. What is Flow Cytometry
     – Definition, Process, Importance
2. What is FACS
     – Definition, Process, Importance
3. What are the Similarities Between Flow Cytometry and FACS
     – Outline of Common Features
4. What is the Difference Between Flow Cytometry and FACS
     – Comparison of Key Differences

Key Terms 

Activated Fluorescence, FACS, Flow Cytometry, Heterogeneous Cell Mixture, Light Scattering

What is Flow Cytometry 

Flow cytometry is an analytical cell biology technique, permitting to determine different parameters of living cells in a heterogeneous mixture. Also, the first impedance-based flow cytometry device was invented by Wallace H. Coulter in 1953. Usually, different parameters measured by flow cytometry include; 

• Cell counting  
• Determining cell characteristics and function 
• Detecting microorganisms 
• Biomarker detection 
• Protein engineering detection 
• Diagnosis of health disorders such as blood cancers 

Process 

Furthermore, the suspension of the sample with several types of cells in fluid allows flowing through the flow cytometer. Inside which, cells ideally flow one cell at a time through a laser beam. Here, cell types show differential light scattering characteristics unique to cell types. Typically, this may be due to the differential expression of proteins, nucleic acid content, intracellular components or cell surface components. 

Figure 1: Flow Cytometry

Analysis 

Besides, in flow cytometry, different types of light scattering patterns and fluorescence emitting patterns involve in the analysis of cell types. They include forward scattered and side scattered light, fluorescence emission, and multiparametric analysis. Of these, forward scattered light refracts from the cell, continuing along the same pathway through which it originally travels. Also, it helps to detect the size of the cell. Meanwhile, side scattered light refracts from the cells in a direction that is outside of the original light path. Thus, it determines the granularity and complexity of different cell types.

Furthermore, after excitation by the compatible wavelength of the laser, fluorescent molecules of cells emit fluorescent light, aiding the identification of different structures of the cells. In addition to that, fluorescent dyes or fluorescence-tagged antibodies can also be used to label specific structures of the cells. Apart from that, forward vs side scattered light is plotted from the populations of leukocytes, aiding the specific identification of granulocytes and lymphocytes. 

What is FACS 

FACS (fluorescence-activated cell sorting) is a modified type of flow cytometry. The main characteristic feature of FACS is its ability to physically separate each cell type in the heterogeneous mixture. For this, this technique uses fluorescently-labeled, target-specific antibodies to identify a particular cell type in the mixture. Hence, FACS separates the heterogeneous cell mixture into two more cell types. Besides, FACS was first invented by Len Herzenberg, who subsequently won the Kyoto Prize in 2006 for his seminal work. 

Figure 2: FACS

Importance 

Moreover, one of the main importance of cell sorting is to separate cells by phenotype. Hence, this allows analyzing the nucleic acid content, protein expression, and metabolic content specific to that phenotype. Secondly, it helps to colony generation of healthy and phenotypically-defined cells for the generation of stem cells or CRISPR cell lines. 

Process 

Usually, in FACS, the sample flows through a vibrating nozzle, which disturbs the stream into droplets, ideally containing one cell. Then, these droplets run through an electrical charging ring, which physically sorts cell types based on their charge.  

Similarities Between Flow Cytometry and FACS 

• Flow cytometry and FACS are two techniques in analytical cell biology. 
• In general, they use fluorescence and other properties to detect different cell types in a heterogeneous mixture. 
• For this, they use differences in cell surface components or intracellular components of different cell types. 
• Also, both methods collect forward-scatter, side-scatter, and fluorescent data. 
• Besides, both techniques have applications in molecular biology, genetics, immunology, pathology, and medicine. 

Difference Between Flow Cytometry and FACS 

Definition 

Flow cytometry refers to the analysis of biological material via detection of the light-absorbing or fluorescing properties of cells or subcellular fractions such as chromosomes as they pass in a narrow stream through a laser beam. Meanwhile, FACS (fluorescence-activated cell sorting) refers to a specialized type of flow cytometry, providing a method for sorting a heterogeneous mixture of biological cells into two or more containers, one cell at a time, based upon the specific light scattering and fluorescent characteristics of each cell. 

Correspondence 

Flow cytometry is an analytical cell biology technique, while FACS is a specialized type of flow cytometry. 

Consequence 

Flow cytometry follows FACS, while FACS is the first step of the analysis of a heterogeneous cell mixture. 

Significance 

Flow cytometry involves the cell analysis and measuring protein expression as a further analyzing technique of FACS, while FACS involves sorting out of cells in a mixed population. 

Function 

Flow cytometry measures the properties of cells such as the number, size, and nucleic acid content of cells, while FACS separates cells into subpopulations from a heterogeneous mixture. 

Sampling Method 

Flow cytometry uses the differential light-scattering properties of cells to collect data, but FACS uses highly specific antibodies tagged with fluorescent dyes to differentiate between cell types.  

Analysis Method 

Flow cytometry uses a sensor to acquire data, but FACS uses an electromagnet to sort the sample. 

Conclusion 

In summary, flow cytometry is an analytical cell biology technique to determine the characteristics of cells in a heterogeneous mixture. Thus, it helps to determine the number of cells, size, and nucleic acid content of the cells, etc. Also, it uses differential light scattering properties of cells unique to each cell type in the mixture. On the other hand, FACS is a type of flow cytometry that permits sorting out of cells in the heterogeneous mixture into two or more types. Also, it uses fluorescence-labeled antibodies to specifically identify components of different cell types. Therefore, FACS is the first analytical technique followed by flow cytometry in protein expression assays. Hence, the main difference between flow cytometry and FACS is the method of cell differentiation and function. 

References:

1. Robinson, Ryan, and Stefan Pellenz. “Ryan Robinson and Stefan Pellenz.” Antibodies, 6 Dec. 2013, Available Here.
2. “Flow Cytometry (FCM) /FACS | Fluorescence-Activated Cell Sorting (FACS).” Sino Biological, Sino Biological Inc., Available Here.

Image Courtesy:

1. “Cytometer” By Kierano – Own work (CC BY 3.0) via Commons Wikimedia   
2. “Fluorescence Assisted Cell Sorting (FACS) B” By SariSabban(CC BY-SA 3.0) via Commons Wikimedia  

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