Fluorescence-Activated Cell Sorting (FACS) is a laboratory technique used to separate and purify distinct cell populations from a complex mixture. This method relies on the unique characteristics of individual cells, allowing researchers and clinicians to isolate specific cells with accuracy. FACS is a valuable tool in various biological research fields and clinical diagnostics, enabling detailed studies of cellular functions and disease mechanisms.
Understanding Flow Cytometry
FACS is an application of flow cytometry, a technology that analyzes individual cells suspended in a fluid stream. In flow cytometry, cells are hydrodynamically focused to pass one at a time through a laser beam. As each cell intercepts the laser, it scatters light, providing information about its physical properties. Forward scatter (FSC) correlates with cell size, while side scatter (SSC) indicates the cell’s internal complexity or granularity.
Flow cytometry also detects fluorescence signals. Cells are often labeled with fluorescent markers that bind to specific molecules on or within them. When these fluorophores are excited by the laser, they emit light at a longer wavelength. This emitted fluorescence reveals the presence and quantity of specific cellular markers. While standard flow cytometry measures these characteristics, FACS extends this capability by physically separating the cells based on these measurements.
The Sorting Mechanism
The feature of FACS is its ability to physically sort cells after analysis. Following optical detection, the fluid stream containing the cells is subjected to high-frequency vibration. This vibration causes the stream to break into individual droplets, with each droplet ideally containing no more than one cell. This ensures that cells are analyzed and sorted on an individual basis.
Based on the fluorescence and light scatter signals detected from each cell, an electrical charge is applied to droplets containing target cells. If a cell expresses a specific fluorescent marker, the system recognizes this signal and applies a positive or negative charge to the droplet enclosing that cell. These charged droplets then pass through an electrostatic deflection system, which consists of oppositely charged plates. The electric field between these plates deflects the charged droplets into different collection tubes, while uncharged droplets containing unwanted cells go to waste. This allows for the isolation of desired cells.
Diverse Applications
FACS is widely applied across scientific disciplines for cell isolation and analysis. In research, it is used to isolate specific cell types for further investigation, such as stem cells for regenerative medicine studies or immune cell subsets to understand immune responses. Researchers also use FACS to analyze cell cycle progression or monitor gene expression within distinct cell populations. The technique facilitates the study of tumor progression and metastasis by isolating specific cancer cell populations for molecular profiling.
In clinical diagnostics, FACS plays a role in identifying and quantifying specific cell populations in patient samples. It is used to diagnose and classify hematological malignancies like leukemia and lymphoma based on the immunophenotype of cells. FACS also monitors disease progression in conditions such as HIV by counting CD4+ T cells. In drug discovery, FACS assists in screening for cells that respond to particular treatments or express certain proteins, aiding in the identification of potential therapeutic targets.
Sample Preparation and Data Interpretation
Accurate results from FACS depend on sample preparation. Cells must be prepared as a homogenous single-cell suspension. This involves dissociation of tissues, followed by filtration. Cells are then stained with fluorescently labeled antibodies or dyes that bind to specific cellular components or surface markers. Maintaining high cell viability is important to minimize background signals.
After processing, the data generated by FACS is analyzed using specialized software. This information is presented as dot plots or histograms, representing the characteristics of individual cells and cell populations. Dot plots can display two parameters simultaneously, such as cell size versus fluorescence intensity, to gate and quantify specific cell subsets. This analysis is important for interpreting complex cellular data and drawing conclusions from FACS experiments.