FiloQuant readouts in comparison to manual analyses. (A) FiloQuant readouts of filopodia number were compared with manual analyses (only the filopodia at the colony edge were considered) in a total of 59 random images of MCF10A and DCIS.COM colonies. Images show examples of MCF10A (left image; no overlay; low filopodia density) and DCIS.COM (right image; fibrillar collagen overlay; high filopodia density) cells migrating collectively in circular invasion assays (imaged using an SDC microscope; 100×, CMOS camera). Insets denote magnified areas, and magenta highlights the filopodial protrusions detected by FiloQuant. Correlation between FiloQuant and manual analyses is displayed on the right. (B and C) FiloQuant readouts of filopodia length were compared with manual analyses in two images of DCIS.COM cells migrating collectively in circular invasion assays (B, no overlay; C, fibrillar collagen overlay). Cells were stained for actin and imaged using an SDC microscope (100×, CMOS camera). Red and yellow insets denote magnified areas, and magenta highlights the filopodial protrusions detected by FiloQuant. Blue arrows point to examples of filopodia that were accurately measured using FiloQuant. Red arrows highlight filopodia that were assigned a shorter length by FiloQuant compared with manual analyses. Discrepancies between FiloQuant readouts and manual measurements are primarily caused by intersecting filopodia (B, red insert), high filopodia density (C, yellow inset), or broken filopodia (C, red inset). Correlation between FiloQuant and manual analyses are displayed on the right, where the blue and red arrows represent the values for the same filopodia indicated in the corresponding images. Bars: (main) 20 µm; (inset) 5 µm.