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 different from flow cytometry in the way that it provides unique characterization versus merely counting and sorting cells.
It is common for the two principles to work in a co-characterization type process to offer a complete qualitative and quantitative approach to flow cytometric analysis. 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 forces 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 with a fluorescent molecule and then added to a mixture of cells. The next step is the process of fluorescence, whereas, specific cells pass through a laser beam they are monitored.
Droplets containing a single cell are assigned a positive or negative charge, based on whether the cell has a fluorescently-tagged antibody. Droplets containing a single cell are then detected by an electric field and diverted into separate collection tubes according to their charge allowing for easy separation of the cells marked with the fluorescent antibody. Multicolor flow cytometry is a useful technique when examining mixed populations of cells, such as blood and tissue cells in human and animal samples.
Generally, a specific cell type is marked with fluorescent dye markers like fluorophore or propidium iodide. The ability to use multiple fluorescent markers simultaneously allows for the identification of multiple cell types, as well as functional markers that further characterize each sample.
There are specialized instruments capable of measuring plus colors 3,4. These fluorescent dyes and markers are measured by different wavelengths of light emitted from the laser to sort by individual cell type.
Each marker is excited at a specific wavelength of light to differentiate them when using multiple markers. When two particles or cells pass through the laser light simultaneously. Or fluorochrome, a fluorescent chemical which emits light after laser excitation.
Fluorescence minus one — specific control, leaving out one of the fluorophores of the complete panel to account for spillover. FSC; scatter coming from the forward direction of the cell reflecting the cells' size. Process of selecting cell populations of interest based on cellular characteristics.
Graph depicting the fluorescence or light scatter intensity with the number of cells on the y-axis. Antibody raised against an antigen not found on the cell of analysis, used to assess non-specific binding. Peripheral blood mononuclear cells, peripheral blood cells consisting of lymphocytes and monocytes.
PMT, a sensitive detector of light at various ranges of the electromagnetic spectrum. SSC; scatter measured at 90 degrees of the laser beam reflecting the granularity or the complexity of the cell. Fluorescent signal of one fluorophore is measured in the channel of another fluorophore, when both are measured on the same cell, resulting in a false positive signal. Flow cytometry: basic principles and applications. Crit Rev Biotechnol.
Trends Immunol. Nolan JP, Condello D. Spectral flow cytometry. Curr Protoc Cytom. Cellular image analysis and imaging by flow cytometry. Clin Lab Med. Compensation in multicolor flow cytometry.
Jolliffe IT, Cadima J. Figure 4. Fluorescent light is filtered so that each PMT detects a specific wavelength. The PMTs convert the energy of a photon into an electronic signal — a voltage. Each PMT will also detect any other fluorochromes emitting at a similar wavelength to the fluorochrome it is detecting. Various filters are used in the flow cytometer to direct photons of the correct wavelength to each PMT Figure 5.
Figure 5. Filters in the flow cytometer. Band pass BP filters allow transmission of photons that have wavelengths within a narrow range. Short pass SP filters allow transmission of photons below a specified wavelength. Long pass LP filters allow transmission of photons above a specified wavelength. Sub heading. As the fluorescing cell passes through the laser beam, it creates a peak or pulse of photon emission over time.
These are detected by the PMT and converted to a voltage pulse, known as an event. The total pulse height and area is measured by the flow cytometer. The measured voltage pulse area will correlate directly to the intensity of fluorescence for that event. Figure 6. The PMT measures the pulse area of the voltage created each time a fluorescing cell releases photons.
When no fluorescing cells pass through the optics, no photons are emitted and no signal is detected. As the fluorescent labeled cell passes through the optics and is interrogated by the laser, photons are emitted and so the intensity of the voltage measured increases. As each fluorescing cell completes its path through the laser beam, this leaves a pulse of voltage over time.
The pulse area is calculated by adding the height values for each time slice of the pulse, determined by the speed of the analog to digital converter ADC , which is 10 MHz i. These events are assigned channels based on pulse intensity pulse area. This signal can be amplified by turning up the voltage going through the PMT. Figure 7. A one parameter histogram plotting channel number vs.
The channels are usually viewed on a log scale on the x axis. Each event is given a channel number depending on its measured intensity; the more intense the fluorescence, the higher the channel number the event is assigned. Figure 8. Fluorescence intensity measurements for a negative and positive result. The negative result shown on the left has no staining and many events at low fluorescence intensity.
A positive result is shown on the right, this has a large number of events at high fluorescence intensity. For a positive result you are looking for the shift in intensity between negative control and a positive samples Figure 9.
Figure 9. Data is from an anonymous Abreview. The ability of a given antibody to resolve a positive signal from a negative signal often depends on which fluorochrome conjugate is used.
This is a general pattern; some differences in the relative intensities are seen for individual antibodies. A highly expressed antigen will usually be detected and resolved from the negative control with almost any fluorochrome. An antigen expressed at lower density might require the higher signal to background ratio provided by a brighter PE or APC conjugate to separate the positive cells adequately from the unstained cells.
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