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Updated: Nov 9, 2025
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Published on: April 4, 2013
An Image-based Dynamic High-throughput Analysis of Adherent Cell Migration
Meng Sun1, Bence Rethi1, Akilan Krishnamurthy1
1Rheumatology Unit, Department of Medicine, Karolinska Insititutet, Stockholm, Sweden.
Abstract:
In this protocol, we describe a method to monitor cell migration by live-cell imaging of adherent cells. Scratching assay is a common method to investigate cell migration or wound healing capacity. However, achieving homogenous scratching, finding the optimal time window for end-point analysis and performing an objective image analysis imply, even for practiced and adept experimenters, a high chance for variability and limited reproducibility. Therefore, our protocol implemented the assessment for cell mobility by using homogenous wound making, sequential imaging and automated image analysis. Cells were cultured in 96-well plates, and after attachment, homogeneous linear scratches were made using the IncuCyte ® WoundMaker. The treatments were added directly to wells and images were captured every 2 hours automatically. Thereafter, the images were processed by defining a scratching mask and a cell confluence mask using a software algorithm. Data analysis was performed using the IncuCyte ® Cell Migration Analysis Software. Thus, our protocol allows a time-lapse analysis of treatment effects on cell migration in a highly reliable, reproducible and re-analyzable manner.
Insights
This protocol enhances cell migration monitoring using live-cell imaging and automated analysis. It offers a reliable and reproducible method for studying cell mobility and treatment effects.
Area of Science:
- Cell Biology
- Biotechnology
Background:
- Scratch assays are standard for studying cell migration and wound healing.
- Traditional scratch assays face challenges in reproducibility due to manual variability in scratching, analysis timing, and image interpretation.
Purpose of the Study:
- To present a refined protocol for monitoring cell migration using live-cell imaging.
- To overcome limitations of conventional scratch assays by implementing homogenous wounding, sequential imaging, and automated analysis.
Main Methods:
- Utilized adherent cells cultured in 96-well plates.
- Performed homogenous linear scratches using the IncuCyte® WoundMaker.
- Acquired sequential images every 2 hours and analyzed using IncuCyte® Cell Migration Analysis Software with automated masking.
Main Results:
- The protocol enables time-lapse analysis of cell migration.
- Automated image analysis ensures objective assessment of cell mobility.
- Demonstrates a highly reliable and reproducible method for studying treatment effects on cell migration.
Conclusions:
- This protocol provides a robust and reproducible method for monitoring cell migration.
- The automated approach minimizes variability, enhancing the reliability of cell migration studies.
- Facilitates accurate, time-lapse assessment of treatment impacts on cell mobility.
