Scratch Assay / Cell Migration Calculator

Calculate cell migration speed, wound closure rate, and relative wound density from scratch assays.

Wound width at t=0 (μm) (μm)

Wound width at t (μm) (μm)

Time elapsed (hours) (hours)

Field width (μm) for density (μm)

Please check your inputs and try again.

Why Rate Per Edge, Not Per Gap

A scratch closes from both sides simultaneously. The gap narrows at twice the speed of either advancing front, so dividing total closure by time gives double the actual migration rate.

Rate per edge = (w₀ − w_t) / (2t)

A wound narrowing from 800 to 200 μm in 24 hours has closed 600 μm, which is 25 μm/hr of gap closure but only 12.5 μm/hr per migrating edge. Reporting the former overstates cell speed twofold.

Metric	Formula	Use
% closure	(w₀ − w_t)/w₀ × 100	Comparing treatments
Rate per edge	(w₀ − w_t)/(2t)	Actual cell speed
Relative to control	treated rate / control rate	Drug effect size

The Proliferation Confound

The scratch assay cannot distinguish cells that moved into the gap from cells that divided into it. Over 24 hours a rapidly proliferating line can close a wound substantially through division alone, and any drug affecting proliferation will appear to affect migration.

The standard control is to include a mitotic inhibitor such as mitomycin C or to serum-starve, so closure reflects migration only. Without it, a scratch assay measuring an antiproliferative compound is uninterpretable.

Confound	Effect	Control
Proliferation	Inflates apparent migration	Mitomycin C or serum starvation
Uneven scratch width	Variable closure rates	Measure multiple positions, use inserts
Debris at wound edge	Blocks migration locally	Wash gently after scratching
Cell death from treatment	Reduces closure, mimics inhibition	Parallel viability assay

The last row matters for drug studies: a compound that kills cells reduces wound closure and looks like a migration inhibitor. A parallel viability assay at the same concentration and timepoint is essential before claiming an effect on motility.

Worked Examples

Example 1: Control: 800→200μm in 24hr

Rate=300/24=12.5μm/hr per edge

Result: 75% closure

Typical epithelial migration speed

Example 2: Drug treated: 800→600μm in 24hr

Rate=100/24=4.2μm/hr per edge

Result: 25% closure — migration inhibited

Anti-migratory compound effect

Example 3: Why proliferation control matters

Fast-dividing line, 24 hr assay, no mitomycin

Result: Closure substantially overstated

A line doubling every 18 hours fills much of the gap by division. Any antiproliferative compound then appears to inhibit migration.

Example 4: Distinguishing toxicity from inhibition

Treated closure 25% of control, viability also 30% of control

Result: Cytotoxicity, not migration inhibition

The cells are dying rather than failing to move. Without the viability data this would be reported as an anti-migratory effect.

Common Mistakes

⚠️

Reporting gap closure as migration rate

The wound closes from both sides, so divide by two. Otherwise cell speed is overstated twofold.

⚠️

Not controlling for proliferation

Dividing cells fill the gap alongside migrating ones. Mitomycin C or serum starvation is needed, especially when testing compounds that affect the cell cycle.

⚠️

Ignoring cytotoxicity

A compound that kills cells reduces closure and mimics migration inhibition. A parallel viability assay is required to separate the two.

⚠️

Measuring at a single position

Scratch width varies along its length. Measure several defined positions or use a culture insert for reproducible gap geometry.

Frequently Asked Questions

Scratch assay limitations?

Cannot distinguish migration from proliferation. Use mitomycin C (2μg/mL 2hr) to arrest proliferation. Quantify: wound width, wound area, wound density. Better: Boyden chamber/transwell for quantitative migration without proliferation confound.

Chemotaxis vs chemokinesis?

Chemotaxis: directed migration toward gradient (Boyden chamber, EZ-TAXIScan). Chemokinesis: undirected increase in migration speed. Scratch assay measures chemokinesis + wound-closure response. Dunn chamber: quantitative chemotaxis in real-time.

Why divide by two when calculating migration rate?

Because the wound closes from both edges simultaneously. Total gap closure is twice the distance any single cell front has travelled.

Why control for proliferation?

Dividing cells fill the gap alongside migrating ones. Without a mitotic inhibitor, the assay measures both processes and cannot separate them.

How do I know a compound inhibits migration rather than killing cells?

Run a parallel viability assay at the same concentration and timepoint. Reduced closure with reduced viability indicates toxicity.

What is the difference between chemotaxis and chemokinesis?

Chemotaxis is directed movement along a gradient; chemokinesis is increased random movement. A scratch assay measures neither cleanly — a Boyden chamber is needed for directed migration.