Fluorescence-activated cell sorting (FACS) is a laboratory technology for identifying and separating specific cells from a larger, mixed group. Imagine trying to find and isolate all the red beads from a massive jar of multicolored beads; FACS automates this process on a microscopic scale. This technique allows scientists to purify populations of cells, even extremely rare ones, from complex biological samples like blood or tissue. By isolating these cells, researchers can study them in detail to unlock information that would be impossible to gather from the original mixture.
The Core Principle of Cell Labeling
The sorting process begins by labeling target cells with fluorescent markers. Many cells have unique surface proteins, known as antigens, that act like cellular name tags. Scientists use specialized molecules called antibodies designed to recognize and bind to these specific antigens. By attaching a fluorescent dye, or fluorophore, to these antibodies, researchers can “paint” the cells they wish to study.
This process is highly specific, as an antibody for one cell type will ignore others. Different fluorophores that glow in various colors can be used simultaneously, allowing for the labeling of multiple characteristics on a single cell or the identification of several cell types in one sample.
The Sorting Process Explained
The journey of a cell through a FACS instrument begins with the fluidics system. The sample of labeled cells is injected into a central core and surrounded by a faster-moving outer stream of a salt-based solution called sheath fluid. This process, known as hydrodynamic focusing, forces the cells to align in a single-file line. This alignment ensures that each cell passes the detection point one at a time for accurate analysis.
As each cell travels in this stream, it passes through one or more focused laser beams at an interrogation point. If a cell is tagged with a fluorescent antibody, the laser excites its fluorophore, causing it to emit colored light. The machine also measures how the cell scatters the laser light. Forward scatter (FSC) is proportional to the cell’s size, while side scatter (SSC) relates to its internal complexity.
Detectors capture these light signals, and optical filters ensure they only measure specific wavelengths from each fluorophore and the scattered light. This data is converted into electronic signals, creating a detailed profile for every cell. This profile includes the cell’s size, complexity, and which fluorescent markers it carries.
The instrument’s stream is vibrated at a high frequency, causing it to break into millions of tiny droplets, each ideally containing one cell. Based on the fluorescence profile, the system applies a positive or negative electrical charge to droplets containing a cell of interest, leaving other droplets uncharged. The stream then passes through an electric field, where charged droplets are deflected into collection tubes while uncharged droplets are discarded.
Applications in Scientific Research and Medicine
In immunology, researchers use FACS to isolate specific immune cells, like helper T-cells or cytotoxic T-cells, from blood. This allows for a better understanding of their function in fighting infections, their role in autoimmune diseases, and their response to vaccines.
Cancer research relies on this technology to isolate rare cancer stem cells from the complex mixture of a tumor. These cells are believed to drive tumor growth and relapse. Studying them provides insight into how cancers resist treatment and spread, which helps guide the development of more effective therapies.
In regenerative medicine, FACS purifies stem cells from sources like bone marrow or fat tissue for research into tissue repair. There is also significant work on using these purified stem cell populations for therapeutic purposes. This includes repairing damaged heart muscle after a heart attack or regenerating bone and cartilage.
Analyzing and Using Sorted Cells
The outcome of the sorting process is a set of tubes containing highly purified populations of living cells. A key advantage of FACS is that the process is gentle enough to keep the cells viable and functional for further experiments.
Once isolated, these cells can be grown in a laboratory dish through a process known as cell culture. This enables researchers to observe their behavior, test their response to drugs, or expand their numbers for analysis. For example, immune cells can be cultured to study how they become activated to fight a pathogen.
Another common use is to analyze the genetic material of the sorted cells. Scientists can extract DNA or RNA from the purified population for detailed genetic sequencing. This can reveal mutations in cancer cells or identify active genes in a specific cell type, a field known as transcriptomics.