The FITC-dextran permeability assay is a method used to measure the integrity of biological barriers. These barriers, formed by cell layers, control the passage of substances into and out of tissues. The assay quantifies the “leakiness” of these barriers, providing a measure of their function and how they are affected by disease or experimental treatments.
Core Components of the Assay
The assay relies on two molecules: fluorescein isothiocyanate (FITC) and dextran. FITC is a fluorescent probe that attaches to other molecules. When exposed to light of a specific wavelength, it emits a bright green light, allowing for precise detection as the amount of light emitted is proportional to the amount of FITC present.
Dextran serves as the tracer molecule that carries the FITC probe. It is a biologically inert polysaccharide, meaning it does not react with or get broken down by the body’s cells. This inertness ensures that its movement across a biological barrier is a passive process, governed by the barrier’s physical properties. Dextrans are available in a wide range of molecular weights, from small 3-4 kilodalton (kDa) molecules to very large 70 kDa or 500 kDa versions.
The choice of dextran size is an important feature of the experimental design. Smaller dextrans, like the 4 kDa version, are used to probe for subtle increases in permeability. They can pass through small gaps between cells, a pathway known as paracellular flux. In contrast, larger dextrans, such as the 70 kDa version, are used to detect more significant damage, as their ability to cross a barrier suggests a substantial loss of integrity.
The Assay Procedure
The FITC-dextran assay is performed using two main experimental models: in vitro (in a lab dish) and in vivo (in a living organism). Each approach provides unique insights into barrier function under different conditions.
The in vitro model uses a Transwell system, which consists of a small insert placed inside a larger well. A porous membrane at the bottom of the insert separates the two compartments. Researchers grow a monolayer of barrier-forming cells, such as intestinal epithelial cells, on this membrane. Once the cell layer is formed, FITC-dextran is added to the top (apical) chamber, and samples are collected from the bottom (basolateral) chamber to measure how much tracer has crossed.
The in vivo model assesses barrier permeability within a living animal, providing a more physiologically relevant context. To study intestinal barrier function, an animal is fasted for a few hours before a solution of FITC-dextran is administered into the stomach via oral gavage. After a set period, a blood sample is collected, and the amount of FITC-dextran that has passed from the intestine into the blood plasma is measured as an indicator of gut permeability.
This in vivo method allows researchers to understand how complex biological systems, including the immune system and gut microbiota, influence barrier integrity. Modifications of this procedure can be used to study other barriers. For example, to assess the blood-brain barrier, FITC-dextran is injected into the bloodstream, and brain tissue is later examined to see if the tracer has leaked from the blood vessels.
Data Acquisition and Interpretation
After the experimental samples are collected, the amount of FITC-dextran that has crossed the barrier is quantified by measuring its fluorescence. A specialized instrument, like a fluorometer or fluorescence microplate reader, is used to expose samples to an excitation light source and measure the emitted light. The reader is set to the specific wavelengths for FITC, typically around 485 nm for excitation and 528 nm for emission.
The raw fluorescence units (RFU) obtained from the reader do not directly represent a concentration. To convert these values into precise measurements, a standard curve must be generated. This involves preparing a series of samples with known concentrations of FITC-dextran. These standards are measured alongside the experimental samples, and their fluorescence readings are plotted against their known concentrations to create a calibration graph.
Using the standard curve, a regression equation is generated, which allows the raw fluorescence value of each experimental sample to be converted into an exact concentration (e.g., ng/mL). A higher concentration of FITC-dextran detected in the collected sample directly corresponds to increased permeability of the barrier being studied. This quantitative data enables researchers to make direct comparisons between different experimental groups.
Applications in Research
The FITC-dextran permeability assay is a widely used tool across biomedical science and translational medicine. Its ability to provide a quantitative measure of barrier function makes it valuable for investigating the mechanisms of various diseases and developing new therapeutic strategies.
In gastroenterology, the assay is used for studying intestinal barrier dysfunction, often called “leaky gut.” It is used to explore conditions like Inflammatory Bowel Disease (IBD) and celiac disease. Researchers can assess how inflammation, diet, or genetic factors contribute to a compromised gut lining, allowing harmful substances to enter the bloodstream.
The assay is also applied in neuroscience to assess the integrity of the blood-brain barrier. This barrier protects the central nervous system, and its breakdown is implicated in neurological disorders like Alzheimer’s disease and multiple sclerosis. By measuring FITC-dextran leakage, scientists can study how these diseases disrupt the barrier and test drugs designed to protect it. The assay also helps investigate systemic barrier failure in conditions like sepsis, where widespread inflammation can cause multiple organ dysfunction.