Fluorescence-activated cell sorting (FACS) 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.
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.
An antibody specific for a particular cell surface protein is associated to a fluorescent molecule and then added to a mixture of cells. For fluorescence when the specific cells pass through a laser beam they are monitored. Droplets containing single cells are given a positive or negative charge, based on whether the cell has limited the fluorescently-tagged antibody or not. Droplets containing a single cell are then detected by an electric field into collection tubes according to their charge.
Interests are first labeled with an antibody which is individual for a particular cell surface molecule. Antibody is coupled to a fluorescent dye, like when in a narrow stream the individual cells pass a laser beam in single file, the fluorescence of each cell is measured. A vibrating nozzle then forms small droplets which each containing a single cell which are given a negative or positive charge based on whether the cell they contain is fluorescing. A strong electric field defects the various charged droplets into separate containers so that each container has a homogeneous population of cells eventually with respect to the cell surface molecule tagged along fluorescent antibody. For biochemical analysis or grown in culture these homogeneous populations may then be used. By flow cytometry the RNA and DNA content of a cell can be measured also.
The cells need to remain viable and without contamination for subsequent culture. See the tips below.
• Include serum in buffers.
• Avoid sodium azide in the buffers during staining as this can be toxic to cells and compromise viability.
• The experiment should be undertaken in aseptic sterile conditions to ensure the cells do not become contaminated.
• It is not usually possible to do intracellular staining before sorting of live cells, as the permeabilization requires damage to the cell membrane which would compromise the cell viability.
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