BenchMasteryMolecular biology techniques & virtual lab

Migration & invasion

A confluent monolayer is scratched with a pipette tip (or a removable insert leaves a defined gap), and the same positions are imaged over time as the edges move in; closure or front speed is quantified from the open area.

% wound closureSimulator readyCore simulatorEngine: Microscopy & imaging
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Scratch / wound healing

A confluent monolayer is scratched with a pipette tip (or a removable insert leaves a defined gap), and the same positions are imaged over time as the edges move in; closure or front speed is quantified from the open area.

How it differs from the standard method

  • Block proliferation (mitomycin C) or use low serum.
  • Make even wounds and wash away debris.
  • Mark positions and image the same fields at every time point.
  • Read while the vehicle wound is still partly open.
In this lesson · about 2 min read
  1. Movement, measured two ways
  2. What the readout actually contains
  3. How these assays mislead
  4. Key terms
  5. When to use it
  6. Sub-techniques

1Movement, measured two ways

Cell migration assays ask how fast cells move and in which direction; invasion assays ask whether they can also get through a barrier of extracellular matrix. The scratch (wound-healing) assay removes a strip of cells from a confluent monolayer and follows the edges as they move back in: collective, two-dimensional migration measured as the fraction of the gap that has closed.

2D, collective

Scratch / wound healing

Cheap and imaging-based. Readout: % closure or front speed (µm/h). Confounded by proliferation and by how even the wound is.

3D-ish, directed

Transwell migration

Cells cross 8 µm pores towards a chemoattractant gradient. Readout: cells per field on the underside.

barrier

Transwell invasion

The same insert coated with Matrigel. Readout: invasion index, invading ÷ migrating cells under the same conditions.

2What the readout actually contains

Closure of a scratch is the sum of cells moving in and cells dividing at the edge. Over a day in serum, a line that doubles in about a day adds a lot of cells, so the assay is usually run in low serum or after blocking division with mitomycin C; otherwise any treatment that slows growth looks like a migration inhibitor.

3How these assays mislead

  • Proliferation counted as migration, in a scratch left in full serum.
  • Wounds of different widths, or different fields imaged at 0 h and later.
  • A wound read after it has closed, or so early that nothing has moved.
  • No gradient across the transwell membrane, or serum in the upper chamber.
  • Unswabbed membranes, overcrowded fields, or reads late enough for crossed cells to divide.
  • Invasion reported without a migration reference.

Key terms

Practise these as flashcards →
Wound closure

1 − open area at time t ÷ open area at 0 h, for the same field.

Chemotaxis

Directed movement up a gradient of a soluble attractant; chemokinesis is a faster but undirected movement.

Mitomycin C

A DNA cross-linker used briefly before a scratch to block cell division so closure reflects migration.

Matrigel

A basement-membrane extract from a mouse sarcoma, rich in laminin and collagen IV, used as the invasion barrier.

Invasion index

Cells crossing a Matrigel-coated insert as a percentage of those crossing an uncoated one, per condition.

Boyden chamber

Two compartments separated by a porous membrane, used to measure migration towards an attractant.

When to use it

  • To compare how fast a monolayer closes a gap under different treatments or after a knockdown (scratch).
  • To test chemotaxis towards a specific factor, with that factor only in the lower chamber (transwell).
  • To ask whether cells can degrade and cross a matrix barrier, and whether that depends on proteases (Matrigel invasion with and without an inhibitor).

Compared with alternatives

Live-cell & time-lapse →

Tracking single cells over time gives speed, persistence and direction per cell rather than a population endpoint.

3D culture & organoids →

Spheroid invasion into a 3D gel is closer to tissue than a 2D scratch or a membrane.

Proliferation & cell cycle →

Check whether a treatment that reduces closure simply slows division.

Image analysis →

Segmenting wound area or counting cells per field reproducibly, with the same settings for every image.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

A confluent monolayer is scratched with a pipette tip (or a removable insert leaves a defined gap), and the same positions are imaged over time as the edges move in; closure or front speed is quantified from the open area.

  • Block proliferation (mitomycin C) or use low serum.
  • Make even wounds and wash away debris.
  • Mark positions and image the same fields at every time point.
  • Read while the vehicle wound is still partly open.