Figure 6. Migration assays for macrophages in 3D. Macrophage infiltration into or migration through 3D matrices can be studied by vertical invasion, spherical invasion, or spheroid gel invasion assays. (A) In a vertical invasion assay, cells are placed on top of a thick matrix. Cells start to infiltrate into the gel (top right, red dots) and can be distinguished from non-invasive cells (black dots), which remain on the gel surface. The proportion of migrating cells, their morphology, and the distance of migration can be quantified over time from bright field microscopy image stacks. Cells can also be imaged by confocal microscopy, as shown by the z-stack reconstruction (bottom right) of human macrophages overexpressing creating a tunnel…white line; overexpressed mCherry-Lifeact (red) is used to stain F-actin. Bar: 20 µm (adapted, with permission, from van Goethem et al.⁵⁷ and Guiet et al.⁶). (B) Spherical invasion assay (SIA). Cells are embedded in a dense plug of type I collagen, which is surrounded by slightly less dense collagen I, containing a chemoattractant. Cells invade into the surrounding matrix and can be monitored by confocal microscopy over time (right panel, bright field images of human macrophages, time of image acquisition after start of experiment is indicated; bar: 10 µm [adapted, with permission from Wiesner et al.⁶²]). The number of infiltrating cells, as well as distance from the plug border, can be quantified. (C) Spheroid gel invasion assay. Macrophages co-cultured for 3 d with spheroids of cancer cells (diameter: 0.5 mm) penetrate into spheroids. Spheroids infiltrated by macrophages are subsequently embedded in Matrigel^TM. Both macrophages and tumor cells leave the spheroid and invade the surrounding matrix. Cells are imaged by multi-photon microscopy (merged micrographs of human macrophages stained with cell tracker [red]; bar: 10 µm). Number of cells moving out of the spheroid (red triangles; bottom scheme) and area of cell invasion (green dotted line) can be quantified (adapted with permission from Guiet et al.⁶). Schemes at the bottom indicate direction of cell movement within each assay.