The scratch assay — also called wound healing assay — is an in vitro cell migration assay in which a cell-free gap is created in a confluent monolayer and the rate of gap closure is measured over time using time-lapse microscopy. It is one of the most widely published methods in cell biology, with applications in cancer biology, drug discovery, toxicology, and wound healing research.
zenCELL owl automates the scratch assay completely: it images all 24 wells every 5–30 minutes inside the CO₂ incubator, calculates gap area, % wound closure, migration rate (µm²/h), and t½ gap closure time automatically — without removing the plate from the incubator at any point.
A scratch assay creates a defined cell-free gap ("wound") in a confluent cell monolayer. Cells at the wound edge migrate into the gap, progressively closing it over 12–72 hours. The rate of closure reflects the migratory capacity of the cells and is quantified as % wound closure per timepoint, migration rate in µm²/h, or t½ gap closure time.
Any adherent cell line that forms a confluent monolayer is compatible. Commonly used cell lines include:
Non-adherent suspension cells cannot be used for scratch assays.
Total assay duration depends on cell line migration speed. Fast-migrating lines (HeLa, A549): 12–24 hours to full closure. Slow-migrating lines (primary fibroblasts, HUVEC): 48–72 hours. Imaging intervals: every 5–30 minutes for complete kinetic data. With zenCELL owl, the assay runs continuously inside the incubator without any manual intervention after plate insertion.
No. The scratch assay runs entirely in brightfield — cell-free wound area is visible without staining, labelling, or fluorescent dyes. A fluorescence microscope is only required if your assay additionally measures a fluorescent reporter (e.g. GFP-tagged protein). For standard migration measurement, brightfield is sufficient and preferred — it eliminates phototoxicity risk.
Pipette tip scratching produces wound width CV of 30–60% across operators — a major source of irreproducibility. ScratchMaker Plates use UV-A photochemical wound creation through a precision mask, achieving CV below 5%. The wound position is fixed relative to the well, eliminating repositioning errors between timepoints. ScratchMaker Plates are compatible with any brightfield microscope and with zenCELL owl.
The primary metric is % wound closure: (A₀ − Aₜ) / A₀ × 100, where A₀ is wound area at T=0 and Aₜ is wound area at timepoint t. Secondary metrics: migration rate (µm²/h) from the slope of the closure curve, and t½ gap closure time. zenCELL owl calculates all metrics automatically. For manual analysis, the free ImageJ Wound Healing Size Tool (Suarez-Arnedo et al., PLoS ONE 2020) is the standard method.
Over 50 peer-reviewed publications document zenCELL owl scratch assay data across oncology, pharmacology, toxicology, and tissue engineering. See the Publications page for a full list.
Place the scratched or ScratchMaker plate inside the CO₂ incubator. zenCELL owl automatically captures T=0 images of all 24 wells simultaneously within minutes of plate insertion. Time-lapse imaging runs continuously at user-defined intervals. The software calculates gap area, % wound closure, migration rate, and t½ for each well at every timepoint. Data exports as CSV. No manual ImageJ analysis required.
| Parameter | zenCELL owl | Manual Microscopy |
|---|---|---|
| Wells imaged simultaneously | 24 | 1 |
| T=0 imaging | All wells simultaneously, <5 min | Sequential, 30–90 min delay |
| Incubator disruption | None | Every timepoint |
| Analysis | Automatic | Manual ImageJ, 5–10 min/well |
| Kinetic resolution | Every 5–30 min, 72h+ | Limited by operator availability |
Book a free demo → See the scratch assay running live in zenCELL owl with real cells.
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Watch zenCELL owl image live inside an incubator. Available.