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Guide to Fluorescence Activated Cell Sorting (FACs): The Latest Technology in Cell Separation


Fluorescence activated cell sorting (FACS) is a cutting-edge technique used to isolate individual cells from a sample and analyse their properties. In this guide, we will discuss the basics of FACS sorting protocol and how it can be used in a variety of applications.

FACS is revolutionizing the field of cell biology and allowing researchers to isolate single cells with an unprecedented accuracy of 99.99%. This makes it an ideal tool for applications where cell purity is important.

How Does Fluorescence Activated Cell Sorting Work ?

Fluorescence activated cell sorting (FACS) is a technique that can be used to isolate specific cell types from a mixed population. FACS is based on the principle that cells can be labeled with fluorescent dyes and sorted according to their fluorescence intensity. FACS is carried out using a flow cytometer, which is a machine that can measure the fluorescence of cells as they pass through a laser beam.

The main benefits of FACS are cell separation, cell labeling, and cell analysis. Cell separation is the most common application of FACS, which is carried out based on a variety of parameters, including cell size, shape, and surface markers. Commonly used surface markers include CD markers such as CD45 (a leukocyte marker), CD14 (a monocyte marker), and HLA-DR (a T-cell marker).

Cell labelling is another common process performed using FACS. Cells can be labeled with fluorescent dyes that bind to specific proteins or DNA sequences. This allows for the detection and quantification of specific proteins or DNA sequences. Cell analysis is the final benefit of FACS. Cells can be analyzed for their size, shape, fluorescence intensity, and other parameters. This allows for the characterization of cell populations.

FACS offers many benefits over traditional cell separation techniques, such as centrifugation and filtration. FACS is much faster than these methods and can often be completed in minutes. Secondly, FACS can be used to purify cells that are difficult to separate using other methods. Finally, FACS can be used to sort cells into multiple groups, allowing for a more detailed analysis of cell populations.

FACs principle

Schematic of the FACs principle.

  1. Cell mixture containing fluorescently labelled cells exits via nozel

  2. Laser beam strikes droplets

  3. FSC detector identifies the size of the cell

  4. SCC detector identifies the granularity or fluorescence of the cell

  5. Electrode assign positive or negative charge

  6. Positive charged cells are drawn to the negative plate while the negative cells are drawn to the poisitive plate

  7. The seperated cells are collected in different collection tubes

Fluorescence Activated Cell Sorting Protocol

Fluorescent activated cell sorting is a complex procedure and requires trained personnel and specialized equipment.

  1. The first step in FACS sorting is to label the cells with fluorescent dyes. The fluorescent dyes are usually attached to antibodies that bind to specific cell surface markers. Cells are routinely labelled with PE or FITC conjugated antibodies. FITC is a green fluorescent dye that is excited by blue light and PE is a red fluorescent dye that is excited by green light. The cells are incubated with the fluorescent dyes for 30-60 minutes.

  2. After incubation, the cells are washed and diluted using a buffer. The cell sorting buffer contains salts, proteins, and other molecules that help to keep the cells healthy during the sorting process. The cells are then resuspended in phosphate buffered saline (PBS) to remove unbound dye.

  3. The next step is to pass the labeled cells through a flow cytometer. The flow cytometer uses lasers to excited the fluorescent dyes and detectors to measure the fluorescence intensity of the dyes. Based on the fluorescence intensity, the flow cytometer sorts the cells into different groups.

  4. The cells are sorted into groups according to their fluorescence intensity and size. The most common sorting method is called forward scatter (FSC) and side scatter (SSC). The FSC channel sorts cells based on their size, with larger cells being sorted into the high FSC channel and smaller cells being sorted into the low FSC channel. The SSC channel sorts cells based on their granularity, with more granular cells being sorted into the high SSC channel and less granular cells being sorted into the low SSC channel.

  5. After sorting, the cell groups are collected in tubes or plates for further analysis. The cell purity can be checked using a microscope or by flow cytometry. Purity can be calculated by dividing the number of cells in the desired group by the total number of cells sorted.

Applications of Fluorescence Activated Cell Sorting

Fluorescence activated cell sorting is a powerful tool that has many applications in biomedical research. Cancer research, immunology research, stem cell research, and developmental biology research are all areas where FACS sorting can be used to isolate specific cell types from a mixed population. FACS is also a valuable tool in identify different subtypes of cells.

Cancer Research

FACS can be used to isolate cancer cells from a mixed population of cells. FACS can also be used to label cancer cells with multiple dyes, allowing for the detection of multiple cell surface markers. This is important because it allows for the identification of different types of cancer cells. FACS can also be used to isolate cancer cells from healthy cells, allowing for the study of specific cancer types.

Immunology Research

FACS can be used to study the immune system. It can be used to isolate specific types of immune cells, such as T-cells and B-cells, from a mixed population. FACS can be used to sort immune cells based on their surface markers. This is important because it allows for the isolation of specific immune cell types that are involved in a particular disease process.

Stem Cell Research

FACS can be used to isolate specific stem cell types from a mixed population. Stem cells can be labelled with fluorescent dyes and sorted according to their fluorescence intensity. This is important because it allows researchers to study the properties of stem cells and develop new treatments for diseases.

Developmental Biology Research

Developmental biology is the study of how organisms develop from fertilization to adulthood. Fluorescence activated cell sorting is a powerful tool that can be used to sort embryonic cells based on their surface markers. This is important because it allows for the isolation of specific embryonic cell types that are involved in a particular stage of development.

In conclusion, FACS has a wide range of applications in both research and clinical settings. In research, FACS is often used to isolate rare cell types such as tumor-infiltrating lymphocytes from cancer patients. In the clinic, FACS is used to diagnose and treat blood disorders, such as leukemia and lymphoma.

Written by Sean Mac Fhearraigh

Seán Mac Fhearraigh PhD is a co-founder of Assay Genie. Seán carried out his undergraduate degree in Genetics at Trinity College Dublin, followed by a PhD at University College Dublin. He carried out a post-doc at the Department of Genetics, University of Cambridge. Seán is now Chief Technical Officer at Assay Genie.


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26th Jul 2022 Seán Mac Fhearraigh, PhD

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