Scratch assay microscopy: A reaction-diffusion equation approach for common instruments and data.

Abstract Scratch assay is an easy and widely used “in vitro” technique to study cell migration and proliferation. In this work we focus on its modelling and on the capability to distinguish between these two phenomena that the simpler and common models are not able to disentangle. We adapted a model based on reaction–diffusion equation for being used with common microscopy instruments/data and therefore taking place in the gap between simpler modelling approaches and complex ones. An optimized image analysis pipeline and numerical least-squares fit provide estimates of the scratch proliferation and diffusion coefficients l and D . This work is intended as a first of a series in which the model is tested and its robustness and reproducibility are evaluated. Test samples were NIH3T3 cells scratch assays with proliferation and migration stimulated by varying the foetal bovine serum amount in the culture medium (10%, 7.5%, 5% and 2.5%). Results demonstrate, notwithstanding an expected l − D anticorrelation, the model capability to disentangle them. The 7.5% serum treatment can be identified as the model sensitivity limit. Treat–control l and D variations showed an intra-experiment reproducibility ( ∼ ± 0 . 05 ∕ h and ∼ ± 200 μ m 2 ∕ h respectively) consistent with single fit typical uncertainties ( ∼ ± 0 . 02 ∕ h and ∼ ± 300 μ m 2 ∕ h respectively).