Transwell Assay
Transwell Migration and Invasion Assay · Also known as: Boyden chamber assay, chemotaxis assay, invasion chamber assay
The Transwell assay (also called the Boyden chamber assay after its originator Stephen Boyden) is a quantitative method for measuring cell migration and invasion in response to chemical gradients or through matrix barriers. The assay uses a membrane insert with defined pore size suspended in a multi-well plate: cells are placed in the upper chamber, a chemoattractant is placed in the lower chamber, and cells that successfully migrate through the pores accumulate in the lower chamber, where they can be counted or visualized. Variants that coat the insert with matrix proteins (Matrigel, collagen) enable measurement of invasion capacity. The Transwell assay is a gold-standard method in cell biology for evaluating cell motility, tumor metastatic potential, and the effects of growth factors and inhibitory compounds.
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When to use it
The Transwell assay is ideal for quantifying directional cell migration in response to chemoattractants, assessing the invasive potential of cancer cells, and evaluating how biomaterial extracts or growth factors affect cell motility. It is preferred when a chemotactic gradient is important (distinguishing chemotaxis from random migration) and when spatial segregation of cell populations is desired. However, the assay requires more setup and incubation time than the scratch assay, is less suitable for real-time or kinetic monitoring, and may be affected by cell sedimentation under gravity. The assay also does not replicate 3D tissue architecture; for complex organotypic models, consider spheroid invasion assays or tissue explant models.
Strengths & limitations
- Quantitative and objective: automated cell counting or luminescence readout minimizes operator bias.
- Measures chemotaxis: the chemical gradient separates directed migration from random motility.
- Versatile: can measure migration alone (uncoated inserts) or invasion (matrix-coated inserts) with the same basic protocol.
- High-throughput capable: 96-well format allows screening of multiple compounds or conditions in parallel.
- Well-characterized: extensive literature and standardized protocols enable cross-study comparisons.
- Requires optimization: optimal incubation time and cell density vary with cell type; pilot studies are needed.
- Does not capture 3D migration: the assay measures movement through defined pores in 2D, not through complex 3D extracellular matrix.
- Labor-intensive: manual staining and counting can be tedious for large numbers of samples; automated imaging systems improve but add cost.
- Cell loss during processing: fixing, staining, and washing can dislodge weakly adhered cells, leading to underestimation of migration.
Frequently asked
What is the optimal incubation time for my cell type?
Incubation time varies with cell type: highly motile cancer cells may require 4–8 hours, while slower fibroblasts or epithelial cells may need 12–24 hours. Perform a kinetic study by harvesting inserts at 4, 8, 12, and 24 hours; plot cell count versus time to identify the linear phase and choose a timepoint in mid-phase.
Should I use serum in the upper chamber, or keep it serum-free?
For measuring chemotaxis to a gradient, use serum-free media in the upper chamber and serum-containing media (or growth factor) in the lower chamber. This creates a gradient driving migration. If using serum in both, you lose directionality. Some applications deliberately use serum in both to measure chemokinesis (random migration) separately.
How do I minimize cell loss during fixation and staining?
Use gentle handling and avoid harsh fixatives if possible. Consider fixing with 4% paraformaldehyde for 10 minutes, then staining. Alternatively, use live-cell dyes (Calcein-AM, DAPI) that do not require fixation and preserve more cells. If using crystal violet, apply carefully and avoid harsh washing.
Can I reuse Transwell inserts after one experiment?
Reusing inserts is not recommended because residual cells, matrix, or stain contaminate the next experiment. Most protocols use disposable inserts. If cost is a concern, perform batch experiments with sufficient replicates per insert to justify single use.
What concentration of Matrigel should I use for invasion assays?
Most protocols use 200–300 µg/mL Matrigel (or 50–100 µL of stock 2 mg/mL per insert), diluted in serum-free media. Optimize coating concentration for your cell type: too much matrix blocks migration completely, while too little provides insufficient resistance. Test concentrations of 100, 200, and 400 µg/mL in a pilot study.
Sources
- Boyden, S. (1962). The chemotactic effect of mixtures of antibody and antigen on polymorphonuclear leucocytes. Journal of Experimental Medicine, 115(3), 453-466. DOI: 10.1084/jem.115.3.453 ↗
- Albini, A., Iwama, Y., Odaka, C., & Bomstein, P. (1987). Exogenous ATIII inhibits basic fibroblast growth factor-induced angiogenesis in vitro. Proceedings of the National Academy of Sciences, 84(17), 6142-6146. link ↗
- Kramer, N., Walzl, A., Unger, C., et al. (2013). In vitro cell migration and invasion assays. Mutation Research/Reviews in Mutation Research, 752(2), 142-195. DOI: 10.1016/j.mrrev.2012.08.001 ↗
How to cite this page
ScholarGate. (2026, June 3). Transwell Migration and Invasion Assay. ScholarGate. https://scholargate.app/en/biomaterials/transwell-assay
Which method?
Set this method beside its closest kin and read them side by side — the library lays the books on the table; the choice is yours.
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