Fluorescence-Activated Cell Sorting, commonly known as FACS, is a laboratory technique used in biological research and medical diagnostics. This technology allows scientists to analyze and separate individual cells from complex mixtures with precision. Its ability to isolate specific cell populations makes it valuable across various scientific disciplines.
Understanding Fluorescence-Activated Cell Sorting
FACS is a specialized application of flow cytometry that integrates its analytical capabilities with the ability to physically sort cells. This method examines individual cells as they pass through a laser beam, assessing their characteristics based on how they scatter light and emit fluorescence. By measuring these properties, FACS can differentiate between various cell types in a sample.
The technology isolates specific cell populations. Researchers tag cells of interest with fluorescent markers that bind to particular cellular components, such as surface proteins or intracellular molecules. This labeling allows the FACS instrument to identify and separate these marked cells from others in a heterogeneous sample, yielding purified populations for further study.
The Process of FACS
The process begins with sample preparation, where cells are suspended in a fluid and often labeled with fluorescent antibodies or dyes. These fluorescent markers attach to specific cellular components, enabling the instrument to identify distinct cell types. The prepared sample is then introduced into the FACS machine’s fluidics system.
Within the fluidics system, cells are hydrodynamically focused into a single-file stream as they pass through a narrow nozzle. This precise alignment ensures that each cell is interrogated individually by a laser beam. As each cell intercepts the laser, it scatters the light in specific patterns and, if fluorescently labeled, emits light at different wavelengths.
Optical detectors capture both the scattered light and the emitted fluorescence signals. Forward scatter (FSC) provides information about cell size, while side scatter (SSC) relates to the cell’s internal complexity or granularity. The fluorescent signals, specific to the markers used, indicate the presence and quantity of particular cellular components. Based on these measurements, the instrument determines whether a cell meets the criteria for sorting.
Following detection, the stream of cells is vibrated at a high frequency, causing it to break into individual droplets. An electrical charge is then applied to droplets containing cells identified for sorting, based on their measured properties. These charged droplets pass through an electric field created by charged deflection plates, which divert them into separate collection tubes. Uncharged droplets, containing unwanted cells, are routed to a waste container.
Where FACS is Used
FACS is used in immunology, allowing researchers to identify and isolate various immune cell subsets, such as T cells, B cells, and macrophages. This capability helps in understanding immune responses, diagnosing immunodeficiencies, and monitoring disease progression. It also aids in the development of cell-based therapies by purifying specific immune cells for adoptive transfer.
In cancer research, FACS is used to detect rare circulating tumor cells, analyze tumor heterogeneity, and study cancer stem cells. By isolating distinct cancer cell populations, scientists can investigate their properties, contributing to the development of targeted therapies and improved diagnostic methods. The technique also helps in assessing the effectiveness of anti-cancer treatments.
FACS is also used in stem cell research, where it purifies specific stem cell populations from mixed samples. This purification helps in understanding stem cell differentiation, self-renewal, and their potential in regenerative medicine. Researchers can isolate pluripotent stem cells or specific progenitor cells for therapeutic applications. The technology also supports drug discovery by enabling high-throughput screening of cell responses to various compounds.