Cell Migration and Invasion Assays: Transwell and Wound Healing

Protocol boundary

Use this as general guidance

Cell-line-specific instructions, assay kit documentation, institutional biosafety requirements, and your lab's validated SOP should take priority when they differ from this general workflow.

Materials

Materials and reagents

Protocol

Step 1: Prepare Cells and Culture Inserts (Transwell)

For a standard Transwell migration experiment, prepare a healthy cell suspension and place cells in the upper chamber of an insert.

An 8 µm pore size is commonly used for many mammalian adherent cell types. Published migration assays frequently use 8 µm inserts with approximately 24-hour migration windows. (PubMed Central (PMC))

Use low-serum or serum-free medium in the upper chamber when appropriate.

Place medium containing a chemoattractant in the lower compartment. Serum gradients are often used; lower-chamber conditions may range from approximately 0.5–10% FBS, depending on cell type and experimental objective.

For an invasion assay, coat the insert membrane with a basement-membrane extracellular matrix hydrogel or another validated ECM barrier before adding cells.

The matrix adds a physical barrier that cells must traverse, distinguishing invasion from simple migration.

Incubation commonly lasts approximately 6–24 hours, although highly motile or slow-moving cells may require different durations.

Protocol

Step 2: Perform Wound Healing (Scratch Assay)

Grow adherent cells to a near-confluent or confluent monolayer.

Create a linear scratch using a sterile pipette tip held as consistently as possible across experimental wells.

Gently wash away detached cells and add the experimental medium.

Capture an image immediately at 0 hours to establish the starting wound area.

Return the cultures to the incubator and image the same regions again, commonly at approximately 24 hours. This 0- and 24-hour design is widely used for scratch assays. (PubMed Central (PMC))

A key limitation is that wound closure can result from both migration and cell proliferation. Low-serum conditions or validated proliferation controls may be necessary when the objective is to specifically measure migration.

Protocol

Step 3: Image and Quantify Migration/Invasion

Transwell Assay

At the endpoint, carefully remove non-migrated cells from the upper membrane surface.

Fix cells that have migrated to the underside and stain them with an appropriate cell stain. Crystal violet is commonly used.

Migrated cells can then be counted across multiple representative microscope fields or quantified using image-analysis software.

Maintain identical imaging rules across experimental groups.

Scratch Assay

Capture images at matched locations and magnification.

A common quantitative measurement is percent wound closure:

• Wound closure (%) = [(initial wound area − final wound area) / initial wound area] × 100

Area-based analysis is usually preferable to measuring only one linear width because scratches may not be perfectly uniform.

Protocol

Step 4: Analyze Data

Normalize results to the appropriate control group.

For Transwell experiments, report migrated or invaded cell number, stained area, or another validated quantitative endpoint.

For wound healing, compare percent area closure between groups.

Include multiple technical fields and independent biological replicates rather than relying on a single image.

Migration and invasion assays should not automatically be treated as equivalent. Increased migration through an uncoated membrane does not necessarily mean that cells can invade through extracellular matrix.

Protocol

Expected Results

In a successful Transwell experiment, migrated cells should be visible on the lower membrane surface while the negative-control condition shows a lower background level.

In scratch assays, the wound area should progressively narrow in motile cultures.

Treatment-dependent differences should be reproducible across independent experiments.

Troubleshooting

Common problems to check

Too Few Migrated Cells

Increase the assay duration within biologically appropriate limits and confirm the chemoattractant gradient and cell viability.

Too Many Cells Cross the Membrane

Reduce starting cell number or assay duration to avoid saturation.

Uneven Scratch Width

Use the same pipette-tip size, pressure, and orientation across wells.

Matrix Detaches During Invasion Assay

Confirm that the ECM layer was prepared and polymerized consistently before cell seeding.

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