FiloQuant can be used to detect filopodia under different cellular contexts and imaging modalities. (A–E) FiloQuant (single image analysis version; software 1) was used to detect filopodia in images acquired from different cell types and using different imaging modalities. (A) DCIS.COM cells migrating collectively were stained for actin and imaged using an SDC microscope (100×, CMOS camera). Bars: (main) 20 µm; (inset) 5 µm. (B) MDA-MB-231 cells transiently expressing mCherry-Myo10 (to visualize filopodia tips) were plated for 2 h on fibronectin (FN), stained for actin, and imaged using a total internal reflection fluorescence (TIRF) microscope. Bars: (main) 20 µm; (inset) 5 µm. (C) A2780 cells transiently expressing mEmerald-LifeAct and migrating on cell-derived matrices in the presence of exogenous FN (labeled with Alexa Fluor 568) were imaged live on an SDC microscope (63× oil objective, EMCCD camera). Bars: (main) 20 µm; (inset) 5 µm. (D) Primary rat hippocampal neurons plated on laminin were fixed, stained for actin and MAP2 (a neuronal marker), and imaged using an SDC microscope (100× objective, CMOS camera). Bars: (main) 20 µm; (inset) 5 µm. (E) NK-92 natural killer cells were seeded on antibody-coated glass (anti-CD18 and anti-NKp30) for 20 min before being fixed, stained for actin, and imaged by TIRF-SIM. The actin-rich filopodia-like protrusions were then detected using FiloQuant (bars, 10 µm). For each panel, FiloQuant analysis was performed on the region of interest defined by the yellow inset, red insets denote magnified areas, and magenta highlights the filopodial protrusions detected by FiloQuant. ROI, region of interest.