A novel approach to quantify the wound closure dynamic

•Cell density can affect the reproducibility and reliability of wound healing assays.•A linear relationship between the wound closure velocity and cell density is observed.•A novel methodology of analysis is proposed to account for cell density effect.•The motility of three populations of MDA-MB-231 breast cancer cells is compared.•YB-1 depleted cells show higher motility, ∆Np63α transfected cells have lower motility.

The Wound Healing (WH) assay is widely used to investigate cell migration in vitro, in order to reach a better understanding of many physiological and pathological phenomena.Several experimental factors, such as uneven cell density among different samples, can affect the reproducibility and reliability of this assay, leading to a discrepancy in the wound closure kinetics among data sets corresponding to the same cell sample. We observed a linear relationship between the wound closure velocity and cell density, and suggested a novel methodological approach, based on transport phenomena concepts, to overcome this source of error on the analysis of the Wound Healing assay. In particular, we propose a simple scaling of the experimental data, based on the interpretation of the wound closure as a diffusion-reaction process. We applied our methodology to the MDA-MB-231 breast cancer cells, whose motility was perturbed by silencing or over-expressing genes involved in the control of cell migration. Our methodological approach leads to a significant improvement in the reproducibility and reliability in the in vitro WH assay.

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