Cell migration is essential for embryonic development, wound healing, and immune responses. Invasion extends migration with the active degradation of extracellular matrix (ECM). These assays are critical in cancer research, angiogenesis studies, and drug screening.

The Scratch (Wound Healing) Assay

The scratch assay is the simplest and most cost-effective migration assay. A confluent monolayer is scratched with a pipette tip to create a cell-free zone, and the rate at which cells migrate into the gap is measured over time.

Protocol:

1. Grow cells to 100% confluence in a 12- or 24-well plate.
2. Serum-starve overnight to suppress proliferation.
3. Scratch the monolayer with a 200 µL pipette tip or a specialized wound-making tool.
4. Wash with PBS to remove detached cells.
5. Add medium with 0.5–2% serum (to minimize proliferation).
6. Image the scratch at intervals (0, 6, 12, 24 hours) using a phase-contrast microscope.
7. Measure the wound area using ImageJ or an automated analysis tool.

The data is expressed as % wound closure or closure rate. The assay is simple but limited by the variability of manual scratches and the confounding effect of proliferation at the wound edge.

Transwell (Boyden Chamber) Assay

The Transwell assay uses a two-chamber system separated by a porous membrane (typically 8 µm pores for most cell types, 3 µm for small cells like lymphocytes). Cells are placed in the upper chamber, and a chemoattractant (e.g., 10% FBS, specific chemokine) is placed in the lower chamber. Cells that migrate through the pores are fixed, stained (crystal violet, DAPI), and counted.

For invasion assays, the upper side of the membrane is coated with a thin layer of Matrigel, a basement membrane extract. Cells must degrade the ECM barrier before migrating through the pores, mimicking physiological invasion more closely than migration alone.

Quantification options:

• Manual counting of stained cells on the membrane underside (5–10 fields per well).
• Solubilizing the stain and measuring absorbance (OD 560–590 nm).
• Pre-labeling cells with Calcein AM and measuring fluorescence of migrated cells.

Real-Time Cell Analysis

Label-free instruments like the xCELLigence use microelectrodes at the bottom of a specialized plate to measure electrical impedance. As cells migrate from the upper well through a membrane, they attach to the microelectrode surface on the lower side, increasing the impedance signal. This provides continuous, real-time migration data without staining or endpoint fixation.

Key Considerations

• Serum gradient: a concentration gradient across the membrane provides directional migration. Without a gradient, random (chemokinetic) movement is measured.
• Matrix coating: for invasion, the membrane pore size, matrix composition (Matrigel, collagen I, fibronectin), and coating thickness all affect results.
• Cell density: too few cells yield poor counts; too many cause aggregation and clogged pores. Optimize density for each cell type.
• Time course: migration through 8 µm pores typically takes 6–24 hours depending on cell type.