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Flow Cytometry and Fluorescence-Activated Cell Sorter (FACS)

by Peter Delves, PhD
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    Flow cytometry is a method for analyzing particles. These could be cells or other types of particles. In this example, we have lymphocytes incubated with a fluorescently tagged monoclonal antibody. Perhaps we want to enumerate the number of CD4+ cells and the number of CD8+ cells in this patient. A sheath fluid containing the antibody labeled cells passes through a small narrow tube where there is just one cell per drop. And laser light is shone onto these cells, passing through one by one down this small tube. One can measure forward light scatter from the laser light which gives a indication of the size of the cell. One can also measure 90 degree light scatter which indicates the granularity of the cell. Red and green fluorescence detectors will detect monoclonal antibodies that are binding to either CD4 or CD8. So one monoclonal antibody against CD4 might be labeled with a dye that emits green fluorescence, and the other antibody against CD8 with a dye that detects red fluorescence. The forward scatter and side scatter allows detection of different cell types. And we can see here an example of the type of plot that the computer linked to the flow cytometer produces. You can see the side scatter on the Y-axis and the forward scatter on the X-axis. And one is able to differentiate neutrophils from monocytes, from lymphocytes. Using monoclonal antibodies, one is able to detect different populations of cells as already mentioned, for example, CD4 and CD8 T-cells. Here we can see the CD4+ T-cells that have been stained with a monoclonal antibody coupled to the diphycoerythrin. And the CD8+ cells detected by a monoclonal antibody coupled to the difluorescein isothiocyanate. The cells that are not staining are mainly B-cells and NK cells because these cell types lack...

    About the Lecture

    The lecture Flow Cytometry and Fluorescence-Activated Cell Sorter (FACS) by Peter Delves, PhD is from the course Immunodiagnostics. It contains the following chapters:

    • Flow Cytometry
    • Fluorescence-Activated Cell Sorter (FACS)

    Included Quiz Questions

    1. Cell granularity
    2. Cell size
    3. Red fluorescence
    4. Green fluorescence
    5. Double positive cells
    1. Monoclonal antibodies
    2. Cell granularity
    3. B cells
    4. Anti-human IgG
    5. Size of the cell
    1. Neutrophils and Basophils
    2. Cells with only one surface marker
    3. Cells with both surface markers
    4. Cells with neither surface markers
    5. Surface markers such as CD4 and CD8
    1. Forward scatter and different types of fluorescence
    2. Cell granularity
    3. Forward scatter and side scatter
    4. Cell surface markers
    5. Monoclonal antibodies

    Author of lecture Flow Cytometry and Fluorescence-Activated Cell Sorter (FACS)

     Peter Delves, PhD

    Peter Delves, PhD


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