How Does Flow Cytometry Sorting Work?
Learn how optical signals from individual cells are used to apply an electrical charge, enabling their precise separation and collection for research.
Flow cytometry sorting is a laboratory technique for identifying and separating specific cells from a mixed population. It is a precise, automated system that inspects individual cells and isolates them based on their unique characteristics. Before separation, the target cells must be tagged and analyzed, providing the information needed for the physical sorting and purification of specific cell types.
Analyzing Cells Before Sorting
The process begins with preparing a sample, such as blood or tissue, into a suspension of individual cells. These cells are labeled using fluorescent markers, like antibodies connected to dyes, which bind to specific proteins known as antigens. This specific binding acts as a “tag,” making the desired cells recognizable by the instrument’s detection systems.
Once labeled, the cell suspension is introduced into the instrument’s fluidics system. This system uses a central fluid stream containing the cells, which is injected into a faster-moving outer fluid called the sheath fluid. This process, known as hydrodynamic focusing, forces the cells to align in a single-file line, ensuring each cell passes through the laser one at a time for precise measurement.
As each cell travels through the instrument, it intersects a focused laser beam, which causes light to scatter and excites any attached fluorescent markers. Detectors measure two types of signals: light scatter and fluorescence. Forward-scattered light provides an approximation of the cell’s size, while side-scattered light indicates its internal complexity. Separate detectors capture the light emitted from the fluorescent tags, identifying which cells have been successfully labeled and converting this data into electronic signals.
The Physical Separation Process
Following the analysis of each cell, the instrument initiates the physical sorting. The fluid stream, containing the single-file cells, is subjected to high-frequency vibration. This vibration causes the stream to break into millions of tiny, uniform droplets. The system is calibrated so that each droplet is likely to contain no more than one cell for effective isolation.
The data gathered from the laser analysis is processed in real-time to identify droplets containing a cell of interest. As a droplet carrying a desired cell forms, the system applies a specific electrical charge to it. Droplets that contain non-target cells, or no cells at all, are left uncharged. This charging decision is made microseconds after the cell passes the laser.
All the droplets then pass through a strong electric field generated by a pair of high-voltage deflection plates. The uncharged droplets are unaffected by the electric field and travel straight into a waste container. The charged droplets are deflected either to the left or right, depending on the polarity of their charge, and guided into separate collection tubes. This results in highly purified populations of the target cells, and some advanced sorters can sort up to four distinct populations simultaneously.
Practical Uses of Sorted Cells
The ability to obtain highly purified cell populations has direct applications in research and medicine. Sorted cells can be cultured in the lab to grow larger populations for experiments or analyzed to understand their biological functions. This purification is valuable when studying rare cells or when analyses require a homogenous sample for accurate results.
In oncology, flow cytometry sorting is used to isolate circulating tumor cells from a patient’s blood. These rare cells can provide information about cancer metastasis and be used to test the effectiveness of different drugs. Researchers in immunology also purify specific immune cells, like T-cells or B-cells, to study their roles in fighting infections, their dysfunction in autoimmune diseases, or their potential for use in immunotherapies.
The technology is also applied in stem cell biology to separate valuable stem cells from other cell types in a tissue sample. These purified stem cells are used in research aimed at developing regenerative medicine therapies. Obtaining a pure population of a single cell type is beneficial for genomic and proteomic studies, as analyzing a uniform group of cells leads to more precise insights into the genetic drivers of cell behavior.