Extended Methods for 2D Confinement.

Physical confinement in microfluidic devices has become a common technique to induce and study cell migration in a large range of cell types. Confined migration was previously understudied due to the limitations of 2D migration assays but has emerged as an important mode of migration in the past decade. Furthermore, confinement improves the quality of the imaging and simplifies the analysis of trajectories by confining migration to the plane of acquisition. Protocols described in this chapter relate to methods extending the previously published 2D confinement technique. First, we explain a method to increase the complexity of the confinement chamber by microfabricating nanometer-sized PDMS grooves on the bottom surface, usually used for contact guidance studies. Then, we describe a method to perform the confinement on cells embedded inside a μm-thin 3D collagen gel. Finally, we describe an alternative method to confine cells based on agarose, so that cells can be fixed or drug perfused while being confined, which is currently not possible in the 2D confinement silicone-based device.