Quantifying Fibroblast Contraction and Elongation

Control or PDPN KD FRCs were seeded at 10,000 per well in 150 μl collagen/matrigel matrix^{8 ,26 (link),28 (link)}. Gels were set at 37°C for 30 minutes then covered with cell culture medium. In some wells, the following were added to both the gel mix and medium: 10 μg/ml CLEC-2-Fc, 10 μM ROCK inhibitor (Y27632) or 10 μg/ml anti-PDPN antibody (R&D, AF3244). Contraction of the gel at day 3 was quantified as the ratio of contracted gel/original area and plotted relative to control. Gels were stained with TRITC-labelled phalloidin and DAPI for maximum length analysis (Imaris). The furthest points of each individual cell were measured in x,y,z coordinates and vectors calculated for comparison (Extended data figure 5).