Background

Directed migration is a key process implicated in physiological and in pathological phenomena, like wound-healing and metastasis. When a cell polarizes and migrates in a directed way, in response to a ligand simulation, it is called chemotactic migration (Stuelten et al., 2018). In adherent and non-adherent cells, migration engages cytoskeletal components and regulators in different manners; chemotactic migration involves close but dissimilar mechanisms, depending on cell types and experimental conditions (Entschladen et al., 2005). To analyse chemotactic migration, various devices have been generated, among which is the classical Boyden chamber assay (transwell assay). In the Boyden chamber assay, cells migrate from the upper to the lower side of a porous membrane, through a gradient of chemoattractant. This assay is valuable for adherent and non-adherent cells, and it offers 3D migration conditions. But its major limitation is that cells can’t be studied while migrating, as with live-cell microscopy. The end results of chemotactic migration in the Boyden chamber are deduced after cell fixation and staining (Chen, 2012). With the development of time-lapse microscopy, based on phase contrast that causes less photodamage, experiments of live-cell imaging for longer periods have become easier to perform. Thus, many 2D random migration devices (Gau and Roy, 2020), and 2D chemotaxis migration devices have been generated (Taylor et al., 2018), such as the ibidi μ-Slide Chemotaxis^® (Zengel et al., 2011). One of the first, and efficient 2D chemotaxis migration assays is the Dunn chemotaxis chamber assay (Zicha et al., 1991). It is a glass chamber, with an inner well free of chemoattractant, and an outer well that contains chemoattractant. Hence, a gradient of chemoattractant is formed on the annular bridge between the two wells. Cells are directly observed while migrating on this bridge, using a time-lapse microscope. A detailed description of the assay is available elsewhere (Monypenny et al., 2009; Chaubey et al., 2011).

Dunn chamber assembly is a multistep process that requires good manual dexterity (Taylor et al., 2018). This might be the reason why it is not frequently used. In this protocol, we will explain how to use the Dunn chamber and how to analyse its results, step by step. The essential contribution of this protocol is that we tried to simplify some steps, in assembly and in analysis, and make them user-friendly, to facilitate the use this device. Finally, we used T47D cells in this protocol (Benseddik et al., 2013). However, any adherent cell line could be used with its respective chemoattractant. Plus, cell morphology and polarity could also be closely observed and analysed within this assay.