Fig. 3

Macrophages (Mϕ) are attracted and orient toward myofibroblasts (MFs) within fibrillar collagen extracellular matrix (ECM). Mϕ within three-dimensional (3D) collagen gel sandwich cultures were tracked over 12 h either a with or b without MFs being present. Mϕ tracks (blue-attracted Mϕ, purple-responsive Mϕ, red-non-responsive Mϕ) were plotted with respect to the MF position or the image center (0,0) (a, 40 Mϕ, 8 experiments; b, 28 Mϕ, 8 experiments). Average local migration velocities were compared between c Mϕ that were attracted and responsive (n = 22) or Mϕ that were non-responsive (n = 28) and d between Mϕ randomly migrating in 2D (n = 41) or 3D collagen (n = 28). Shown are medians (center lines) of box (25th to 75th percentiles) and whisker (minimum to maximum) plots. e Mϕ and MFs were co-cultured within 3D collagen gel sandwich cultures for 3 h, followed by staining for F-actin (Phalloidin, green), Mϕ marker (F4/80, red) and nuclei (DAPI, blue). Integrated F-actin fluorescence intensity profiles along lines drawn from the Mϕ centroid in 1° angle distances (shown for every 5°) over 360° were used to identify the leading (highest actin content). Zero angle was defined by a line connecting Mϕ and MF centroids. f Three additional examples for Mϕ orientations toward MFs in the same ECM. g Peak angles from e were summarized to relate angle orientation of Mϕ to the position of MFs (0°) in rose plots. Mϕ with orientation angles <60° were considered to have polar-directed orientation toward MFs (n = 57). All scale bars: 25 µm