A simple 3D cellular chemotaxis assay and analysis workflow suitable for a wide range of migrating cells.

Cellular migration plays a crucial role within multicellular organisms enabling organ development, wound healing and efficient immune responses, but also metastasis. Therefore, it is crucial to dissect the underlying mechanisms. Directed migration and invasion are most efficient in response to chemotactic signals. To study cell migration and chemotactic responses, current experimental setups use either simplified 2D, tissue-mimetic 3D (e.g. collagen matrices) or in vivo environments. While the in vivo experiments are closest to the real physiological situation, they require animal experiments and are thus ill-suited for screening purposes. 3D matrices, on the other hand, can mimic in vivo conditions in many respects thus serving as instructive settings for the initial dissection of cell migration and chemotaxis. However, performing 3D chemotaxis assays has its limitations due to the delicate nature of most available setups and the tedious and time-consuming manual quantification process. Here, we present •A method for the easy construction of a chemotaxis chamber suitable for the analysis of large cell numbers.•A procedure to quantify their migration automatically with minimal input required by the experimenter.•Both successfully validated by analyzing the 3D chemotaxis of highly migratory primary dendritic cells and the invasive MDA-MB-231 cancer cells.