Abstract
The scratch assay is an in vitro technique used to assess the contribution of molecular and cellular mechanisms to cell migration. The assay can also be used to evaluate therapeutic compounds before clinical use. Current quantification methods of scratch assays deal poorly with irregular cell-free areas and crooked leading edges which are features typically present in the experimental data. We introduce a new migration quantification method, called 'monolayer edge velocimetry', that permits analysis of low-quality experimental data and better statistical classification of migration rates than standard quantification methods. The new method relies on quantifying the horizontal component of the cell monolayer velocity across the leading edge. By performing a classification test on in silico data, we show that the method exhibits significantly lower statistical errors than standard methods. When applied to in vitro data, our method outperforms standard methods by detecting differences in the migration rates between different cell groups that the other methods could not detect. Application of this new method will enable quantification of migration rates from in vitro scratch assay data that cannot be analysed using existing methods.