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    Facs

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    Manïkandan Sridhar
    Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry that allows for the sorting of cells into containers based on their light scattering and fluorescent characteristics. Cells are labeled with fluorescent antibodies and passed in a stream through a laser, where their fluorescence is measured. Droplets are then charged based on fluorescence and deflected electrostatically into separate containers. FACS provides a fast, objective way to separate cells based on cell surface markers and analyze fluorescent signals from individual cells.

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    Facs

    Uploaded by

    Manïkandan Sridhar
    2K views15 pages
    Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry that allows for the sorting of cells into containers based on their light scattering and fluorescent characteristics. Cells are labeled with fluorescent antibodies and passed in a stream through a laser, where their fluorescence is measured. Droplets are then charged based on fluorescence and deflected electrostatically into separate containers. FACS provides a fast, objective way to separate cells based on cell surface markers and analyze fluorescent signals from individual cells.

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    Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry that allows for the sorting of cells into containers based on their light scattering and fluorescent characteristics. Cells are labeled with fluorescent antibodies and passed in a stream through a laser, where their fluorescence is measured. Droplets are then charged based on fluorescence and deflected electrostatically into separate containers. FACS provides a fast, objective way to separate cells based on cell surface markers and analyze fluorescent signals from individual cells.

    Copyright:

    Attribution Non-Commercial (BY-NC)
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    Download as ppt, pdf, or txt
    2K views15 pages

    Facs

    Uploaded by

    Manïkandan Sridhar
    Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry that allows for the sorting of cells into containers based on their light scattering and fluorescent characteristics. Cells are labeled with fluorescent antibodies and passed in a stream through a laser, where their fluorescence is measured. Droplets are then charged based on fluorescence and deflected electrostatically into separate containers. FACS provides a fast, objective way to separate cells based on cell surface markers and analyze fluorescent signals from individual cells.

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    Fluorescence-activated cell sorting (FACS) is a specialized type of flow cytometry that allows for the separation and sorting of cells into containers based on their specific light scattering and fluorescent characteristics. It was developed in 1972 by Leonard Herzenberg at Stanford University and involves staining cells with fluorescent antibodies and using a flow cytometer to sort the cells based on their fluorescent properties.

    Fluorescence-activated cell sorting (FACS) uses flow cytometry to sort cells that have been treated with fluorescent antibodies or dyes. Cells pass through a flow cytometer one by one, where they are exposed to laser light and optical sensors measure the light scattered and emitted by each cell. Based on these measurements, a charge is applied to each cell droplet as it breaks off from the stream. The charged droplets are then deflected into different containers by electric fields.

    Some applications of flow cytometry include immunofluorescence, cell cycle analysis, cell kinetics studies, genetics and molecular biology research, microbiology, biological oceanography, parasitology, hematology, oncology, and transplantation medicine.

    FLUORESCENCE

    ACTIVATED CELL SORTING

    S.SHRIHARI
    (21107214087)
    DEFINITION
    Fluorescence-activated cell sorting is a specialized type of flow cytometry. It
    provides 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. It is a useful
    scientific instrument, as it provides fast, objective and quantitative recording of
    fluorescent signals from individual cells as well as physical separation of cells of
    particular interest.

    FACS is a trademark of Becton Dickinson Immunocytometry Systems


    (BDIS). All FACS instruments are BDIS systems, but not all cytometers
    are FACS.
    History
    In 1972 L. Herzenberg (Stanford Univ.), developed a cell sorter that
    separated cells stained with fluorescent antibodies. The Herzenberg
    group coined the term.
    PRINCIPLE

    The cell suspension is entrained in the center of a narrow, rapidly flowing


    stream of liquid. The flow is arranged so that there is a large separation
    between cells relative to their diameter. A vibrating mechanism causes the
    stream of cells to break into individual droplets. The system is adjusted so
    that there is a low probability of more than one cell per droplet. Just before
    the stream breaks into droplets, the flow passes through a fluorescence
    measuring station where the fluorescent character of interest of each cell is
    measured. An electrical charging ring is placed just at the point where the
    stream breaks into droplets. A charge is placed on the ring based on the
    immediately-prior fluorescence intensity measurement, and the opposite
    charge is trapped on the droplet as it breaks from the stream. The charged
    droplets then fall through an electrostatic deflection system that diverts
    droplets into containers based upon their charge. In some systems, the charge
    is applied directly to the stream, and the droplet breaking off retains charge of
    the same sign as the stream. The stream is then returned to neutral after the
    droplet breaks off.
    Fluorescence Activation Process
    (or Immunofluorescence)
    Antibodies recognize specific Antibodies are artificially conjugated to
    molecules in the surface of some cells
    fluorochromes.
    FITC FITC When the cells are analyzed by flow cytometry
    Antibodies
    the cells expressing the marker for which the
    antibody is specific will manifest fluorescence.
    Cells who lack the marker will not manifest
    fluorescence.
    FITC

    FITC
    ITC
    F

    C
    FIT

    But not
    others
    Flow Cytometry Applications
    APPLICATIONS
     Immunofluorescence
     Cell Cycle Kinetics
     Cell Kinetics
     Genetics
     Molecular Biology
     Animal Husbandry (and Human as well)
     Microbiology
     Biological Oceanography
     Parasitology
     Bioterrorism
    MEDICINE

    Transplantation, Hematology, Tumor


    immunology and Chemotherapy, Genetics and S
    perm sorting for Sex preselection
    Cells are
    Z Y presented to the
    Sample
    laser using
    Sheath
    principles of
    X hydrodynamic
    focusing

    Flow
    chamber

    Y Z

    Laser optics X

    Laser Beam
    Gating and Statistics

    • Data generated in flow cytometry is displayed using


    Multiparamater Acquisition and Display software
    platforms.
    • Histograms corresponding to each of the
    parameters of interest can be analyzed using
    statistical tools to calculate percentage of cells
    manifesting specific fluorescence, and fluorescence
    intensity.
    • This information can be used to look at
    fluorescence expression within subpopulations of
    cells in a sample (gating).
    Flow Cytometry Data

    Smaller
    Region,
    Live cells
    mostly

    Larger Region
    includes all
    cells
    Running Samples

    • Prepare samples.
    • One sample should be completely negative. This
    sample should be analyzed first. This sample is
    used for adjusting the PMT’s amplification voltage.
    • Adjust the PMT Voltage until you can see a
    population peak in the first decade of your 1
    parameter and or your two parameter plot.These
    samples are used for adjusting Spectral Overlap.
    • Once the instrument settings are optimized, run
    samples and collect data.
    Hydrodynamic Systems
    Sample in
    Sheath

    Piezoelectric
    crystal oscillator

    Sheath in

    Signal Fluorescence
    direction Sensors

    Laser beam
    Scatter Sensor

    Sheath Core

    ++ --

    Flow Chamber
    Light Scatter
    • Materials scatter light at wavelengths at which they do not absorb.
    • If we consider the visible spectrum to be 350-850 nm then small particles
    (< 1/10 λ ) scatter rather than absorb light.
    • For small particles (molecular up to sub micron) the Rayleigh scatter
    intensity at 0o and 180o are about the same.
    • For larger particles (i.e. size from 1/4 to tens of wavelengths) larger
    amounts of scatter occur in the forward not the side scatter direction - this
    is called Mie Scatter (after Gustav Mie) - thus forward scatter is related to
    size (at 1-15 microns).

    Shapiro
    Shapiropp79
    79
    Frequency distribution

    histogram
    Number of events

    Intensity of parameter (e.g. fluorescence)


    Fluorescence Activated
    Cell Sorting
    488 nm laser FALS Sensor

    Fluorescence detector

    - +
    Charged Plates

    Single cells sorted


    into test tubes
    Sample in Sheath

    Stream
    Charge Piezoelectric
    crystal oscillator

    Sheath in
    Sensors

    Laser beam Sensor


    SORT DECISIONS
    SMALL BEAD LARGE BEAD

    Last attached
    droplet
    SORT LEFT SORT RIGHT

    +2KV -2KV
    Cell sorting LEFT RIGHT Frequency Histogram

    Waste
    SMALL BEAD LARGE BEAD

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