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Deep Dive into the Transwell Migration and Invasion Assay
- CLYTE research team
- Oct 6
- 8 min read
The intricate dance of cell movement, a fundamental process in life, governs everything from tissue development and wound healing to the devastating spread of cancer. To understand these cellular ballets, scientists rely on powerful laboratory techniques, and among the most prominent is the Transwell migration and invasion assay. This versatile and widely used in vitro tool provides a window into the migratory and invasive capabilities of cells, offering crucial insights into a multitude of biological processes. This article will guide you through the principles, protocols, and applications of the Transwell assay, providing a comprehensive overview of this essential research technique.
The Transwell Assay: A Tale of Two Chambers
At its core, the Transwell assay, also known as the Boyden chamber assay, is a beautifully simple yet powerful system. It utilizes a specialized cell culture insert that fits snugly into the well of a larger plate, effectively creating two distinct chambers: an upper and a lower chamber, separated by a porous membrane.
The Upper Chamber: This is where the cells of interest are seeded.
The Lower Chamber: This chamber is filled with a medium that may contain a chemoattractant—a substance that attracts cells and stimulates their movement.
The Porous Membrane: This is the heart of the assay. The membrane is peppered with microscopic pores of a specific size, which are large enough for cells to actively squeeze through but small enough to prevent passive movement.
The fundamental principle of the assay lies in the creation of a chemoattractant gradient. The higher concentration of the chemoattractant in the lower chamber coaxes the cells in the upper chamber to migrate through the pores of the membrane towards the stimulus.
Migration vs. Invasion: What's the Difference?
The Transwell assay can be adapted to study two distinct but related cellular processes:
Migration Assay: This assay measures the ability of cells to move through the porous membrane in response to a chemoattractant. It assesses the cells' intrinsic migratory capacity, or chemotaxis.
Invasion Assay: To mimic the complex environment cells encounter in vivo, the invasion assay takes the migration assay a step further. In this setup, the porous membrane is coated with a layer of extracellular matrix (ECM) proteins, such as Matrigel. This ECM layer acts as a barrier that cells must actively degrade and penetrate to reach the chemoattractant in the lower chamber. The invasion assay, therefore, measures a cell's ability to not only migrate but also to invade through a biological barrier, a hallmark of cancer metastasis.
A Step-by-Step Guide to the Transwell Assay Protocol
Phase 1: Preparation and Setup
This initial phase is critical for ensuring the assay runs smoothly and yields reliable data.
Rehydrate the Inserts: The porous membranes of the Transwell inserts are typically shipped dry. Before use, they must be rehydrated. This is done by adding warm, serum-free cell culture medium to both the upper and lower chambers and allowing them to incubate, usually for 1-2 hours in a cell culture incubator (37°C, 5% CO₂). This ensures the pores are open and ready for cell passage.
Prepare the Chemoattractant: The lower chamber is filled with the medium that will attract the cells. This is often a complete medium containing a higher concentration of a chemoattractant, such as fetal bovine serum (FBS), or specific growth factors like VEGF or PDGF. The upper chamber will contain a medium with a lower concentration of the attractant (or none at all) to establish the necessary chemical gradient.
Coat for Invasion Assays (If Applicable): If you are performing an invasion assay, this is the stage where you create the barrier.
A thin layer of a basement membrane extract, most commonly Matrigel, is carefully applied to the top surface of the rehydrated membrane.
This gel is a mixture of extracellular matrix (ECM) proteins that solidifies to form a barrier.
The plate is then incubated to allow the gel to polymerize. This step mimics the in vivo environment where cells must degrade the ECM to move through tissue. For a migration assay, this coating step is skipped.
Phase 2: Cell Preparation and Seeding
Proper handling of the cells before the assay is crucial for a clean result.
Harvest and Count Cells: The cells you plan to study are grown in standard culture flasks. They are harvested using trypsin, washed to remove any residual serum, and then counted using a hemocytometer or automated cell counter to determine their concentration.
Serum Starvation (Optional but Recommended): Often, cells are "serum-starved" for several hours (e.g., 4-24 hours) before the assay. This involves culturing them in a medium without serum. This step reduces baseline cell motility and makes the cells more responsive to the chemoattractant you introduce during the assay, leading to a stronger and clearer signal.
Resuspend and Seed: The cells are resuspended in a low-serum or serum-free medium at a specific concentration. A precise volume of this cell suspension is then carefully pipetted into the upper chamber of the prepared Transwell insert. It's important to avoid introducing air bubbles.
Phase 3: Incubation
This is the active phase where cell movement occurs.
Assemble the Chambers: The Transwell insert (containing the cells in the upper chamber) is gently placed into the well of the culture plate (containing the chemoattractant in the lower chamber).
Incubate: The entire plate is placed in a cell culture incubator. The incubation time is a critical variable and depends on the cell type's motility. It can range from as short as 4 hours for highly motile cells to 48 hours for slower ones. The goal is to allow enough time for a significant number of cells to migrate without allowing enough time for cells to start dividing, which would confound the results.
Phase 4: Post-Incubation Processing
After incubation, you must process the inserts to visualize and count only the cells that successfully moved through the membrane.
Remove Non-Migrated Cells: The first step is to get rid of the cells that did not migrate and are still in the upper chamber. This is typically done by gently inserting a moist cotton swab into the insert and carefully wiping away the cells and any ECM gel (in an invasion assay).
Fixation: The insert is then moved to a new well containing a fixative solution, such as 4% paraformaldehyde or methanol. This process kills the cells but preserves their morphology and adheres them firmly to the underside of the membrane.
Staining: After fixation, the cells are stained to make them visible under a microscope. A common and simple stain is crystal violet, which is a deep purple dye that vividly stains the cell nuclei and cytoplasm. The insert is submerged in the staining solution for about 15-30 minutes and then washed several times in water to remove excess stain.
Phase 5: Analysis and Quantification
The final step is to count the migrated or invaded cells to get a quantitative result.
Imaging: The stained membrane is allowed to dry completely. It can then be visualized using a light microscope. Sometimes, the membrane is carefully cut out of the insert with a scalpel and mounted onto a microscope slide.
Counting and Quantification: The number of stained cells on the underside of the membrane is counted.
Manual Counting: The researcher typically counts the cells in several different fields of view under the microscope and calculates an average.
Automated Counting: Alternatively, images can be taken of each membrane and analyzed using software like ImageJ, which can automatically count the stained cells.
Extraction Method: Another method involves dissolving the crystal violet stain from the cells using a solvent (like acetic acid or methanol) and then measuring the absorbance of the resulting solution with a spectrophotometer. The higher the absorbance, the more cells were present.
The final count provides a quantitative measure of the cells' migratory or invasive capacity in response to the specific chemoattractant used.
Optimizing Your Transwell Assay for Success
To ensure reliable and reproducible results, it's crucial to optimize the parameters of your Transwell assay. Key considerations include:
Pore Size: The choice of membrane pore size is critical and depends on the size and deformability of the cells being studied.
Cell Seeding Density: The number of cells seeded in the upper chamber can significantly impact the results.
Chemoattractant Concentration: The concentration of the chemoattractant should be optimized to elicit a robust migratory or invasive response.
Incubation Time: The incubation time needs to be long enough to allow for detectable cell migration or invasion but not so long that cell proliferation confounds the results.
Applications of the Transwell Assay: From Basic Research to Drug Discovery
The Transwell migration and invasion assay is a workhorse in many areas of biological research, including:
Cancer Research: Studying the metastatic potential of cancer cells and screening for anti-cancer drugs that inhibit cell migration and invasion.
Immunology: Investigating the migration of immune cells in response to inflammatory signals.
Developmental Biology: Understanding the role of cell migration in embryonic development and tissue formation.
Wound Healing: Studying the migration of fibroblasts and other cells involved in the wound healing process.
By providing a quantitative and reproducible method to study cell movement, the Transwell assay continues to be an indispensable tool for unraveling the complexities of cell biology and disease.
Frequently Asked Questions: Transwell migration and invasion assay
What is a Transwell invasion assay?
A Transwell invasion assay is a specific type of in vitro (in the lab) experiment designed to measure a cell's ability to move through a barrier that mimics the natural environment of tissues. It uses a two-chamber system called a Transwell insert, where the upper and lower chambers are separated by a porous membrane. For an invasion assay, this membrane is coated with a layer of extracellular matrix (ECM) proteins, such as Matrigel.
To invade, cells seeded in the upper chamber must actively degrade and penetrate this ECM barrier to reach a chemoattractant (a substance that attracts cells, like serum) in the lower chamber. This process simulates the invasive behavior of cells, which is a critical characteristic of cancer metastasis.
How do you do a Transwell migration assay?
Performing a Transwell migration assay involves a precise, multi-step process:
Preparation: First, you rehydrate the Transwell insert's porous membrane with serum-free medium. Then, you add a medium containing a chemoattractant to the lower chamber.
Cell Seeding: Next, you prepare your cells. This often involves serum-starving them to increase their responsiveness. You then count the cells and add a specific number into the upper chamber in a serum-free medium.
Incubation: The plate is placed in an incubator (typically 37°C, 5% CO₂) for a set period (e.g., 4-48 hours), allowing the cells to migrate through the pores toward the chemoattractant.
Processing: After incubation, you remove the non-migrated cells from the top of the membrane with a cotton swab. The cells that successfully migrated to the bottom of the membrane are then fixed (to preserve them) and stained (usually with crystal violet) to make them visible.
Analysis: Finally, you count the stained cells on the bottom of the membrane using a microscope. This count gives you a quantitative measure of cell migration.
What is the difference between a migration assay and an invasion assay?
The key difference between a migration assay and an invasion assay lies in the presence of an extracellular matrix (ECM) barrier.
Migration Assay: This assay measures a cell's intrinsic ability to move through the pores of a membrane in response to a chemical signal (chemotaxis). The membrane is uncoated. It answers the question: "Can the cells move?"
Invasion Assay: This assay measures a cell's ability to not only move but also to actively degrade and penetrate a biological barrier. The membrane is coated with a layer of ECM proteins (like Matrigel). It answers the more complex question: "Can the cells degrade a barrier and then move?"
Essentially, invasion is a specialized form of migration that requires an extra, more aggressive step.
How do you analyze a Transwell assay?
Analyzing a Transwell assay involves quantifying the number of cells that have successfully moved to the bottom of the membrane. There are several common methods to do this:
Manual Counting: This is the most direct method. After staining, you place the membrane under a microscope and manually count the number of cells in several different fields of view. You then calculate the average number of cells per field to get a representative count for the entire membrane.
Image-Based Analysis: You can capture images of different fields of view and use software like ImageJ to automatically count the cells. This method is often faster and less prone to user bias than manual counting.
Stain Elution and Spectrophotometry: In this method, after staining with crystal violet, you use a solvent (like acetic acid) to dissolve the dye from the cells. You then transfer this colored solution to a plate reader and measure its absorbance. The absorbance value is directly proportional to the number of cells, allowing for high-throughput quantification.
Regardless of the method, the final data is typically presented as the number or percentage of migrated/invaded cells relative to a control group, allowing for statistical comparison between different experimental conditions.
