Cell sorting flow cytometry is a powerful laboratory technique used to analyze and physically separate specific cells from a heterogeneous mixture. This process, often referred to as Fluorescence-Activated Cell Sorting (FACS), allows researchers to isolate a pure population of cells based on their physical and fluorescent characteristics. The ability to purify cells at the single-cell level is instrumental in modern biological research, particularly for experiments that require high purity for subsequent downstream analysis. Cell sorting is a preparative tool, providing a homogeneous sample for further study or therapeutic applications.
Understanding the Technology
The flow cytometer is an instrument that analyzes cells before the sorting process begins, relying on three core technological components: fluidics, optics, and electronics.
The fluidics system precisely aligns the cells from the sample tube into a single file within a stream of sheath fluid, a process known as hydrodynamic focusing. This alignment ensures that each cell passes individually through the laser interrogation point, which is necessary for accurate measurement.
The optical system contains one or more lasers that excite the cells as they pass through the focused beam. Cells naturally scatter the laser light, providing two types of information: forward scatter (FSC), which relates to cell size, and side scatter (SSC), which relates to the internal complexity or granularity of the cell. If cells are labeled with fluorescent tags, the laser causes these tags to emit light at different wavelengths.
The detectors capture this scattered light and fluorescence, converting the signals into proportional electronic signals. The electronics system amplifies these analog signals and converts them into digital data points for computer processing. This data allows researchers to distinguish different cell populations based on their unique light scatter and fluorescence profiles, which is the foundation for determining which cells should be separated.
The Cell Sorting Process
The physical mechanism of separation in a cell sorter is based on the principle of electrostatic deflection, triggered after the electronic system identifies a cell of interest.
The fluid stream carrying the cells is forced through a tiny nozzle vibrated at a high frequency by a piezoelectric crystal. This vibration causes the continuous stream of liquid to break up into a stable cascade of uniform, individual droplets.
The moment a cell is analyzed is separated by a time delay, known as the drop delay, from the point where the stream breaks into droplets. This delay is precisely calculated to ensure the cell of interest is encapsulated within a single droplet. Once the electronics identify a target cell, a computer-controlled system applies an electrical charge to the stream just before the droplet containing the target cell breaks off.
The charged droplet separates from the rest of the stream and travels between a pair of high-voltage deflection plates. The droplet is attracted toward the plate with the opposite charge, causing its trajectory to be physically deflected into a designated collection tube. Droplets containing unwanted cells remain uncharged and pass straight into a central waste collector, resulting in a highly purified population.
Major Uses in Science and Medicine
Cell sorting is an indispensable tool across numerous scientific and medical fields due to its ability to isolate pure cell populations with high precision. This capability is crucial for subsequent downstream analysis and therapeutic development.
In immunology, researchers routinely use the technique to isolate specific subsets of immune cells, such as T-lymphocytes and B-lymphocytes, based on their distinct surface proteins. Purifying these populations allows for detailed studies of their function, development, and role in disease response.
Cell sorting is also utilized in cancer diagnostics and research for enriching rare cell types from complex samples. Circulating tumor cells (CTCs), shed from a primary tumor, can be isolated despite their extremely low frequency in the blood. Isolating CTCs provides material for molecular profiling, aiding in understanding tumor progression and developing targeted therapies.
The technique is fundamental in genetic studies, used to isolate fluorescently tagged cells, such as those that have successfully taken up a gene in a gene therapy experiment. This allows scientists to obtain a homogeneous population of genetically modified cells for expansion or analysis. Sorting also purifies progenitor cells in stem cell research for use in regenerative medicine.
Post-Sorting Cell Viability
Post-sorting cell viability is a major consideration, as the utility of the output depends on the quality and integrity of the cells. Since many downstream applications require live, healthy cells, the sorting process must be gentle enough to maintain cellular health. Factors like the pressure used to drive the fluidics and the physical forces involved in droplet formation can induce stress.
To preserve cell viability, sorters often use larger nozzle diameters, such as 100 micrometers, which allow for lower sheath pressures and minimize mechanical stress. The temperature of the sample and the collection buffer is also carefully controlled, often recommending 4°C to slow down cellular metabolism. The collection medium is typically enriched with proteins and buffering agents to maintain the cell’s physiological environment.