Figure 6.
Figure 6. Filopodia dynamics can be analyzed using FiloQuant in circular invasion assays. (A and B) MCF10A and DCIS.COM cells stably expressing LifeAct-mRFP were plated in circular invasion assays and left to invade through fibrillar collagen I or GFR Matrigel for 3 d before being imaged live using an SDC microscope (100× objective, EMCCD camera) for over 9 h (one picture every 3 min). Filopodia were then automatically detected using the automated version of FiloQuant (software 3) and tracked using TrackMate (ImageJ-based tracking tool). For each condition, stills of single z planes with filopodia detected by FiloQuant (magenta) are displayed (bar, 25 µm) at different time points. In addition, time projections of detected filopodia as well as filopodia tracks are shown. The time projections were directly generated by FiloQuant and are color coded as a function of time. For filopodia tracking, tracking files, generated by FiloQuant, were entered into TrackMate for automated tracking. Filopodia tracks generated by TrackMate are color coded as a function of the track starting time (track index; A). For each condition, the percentage of stable filopodia (lifetime > 3 min) and the mean lifetime of stable filopodia were quantified from two independent experiments and displayed as Tukey box plots (B; ***, P < 2.79 × 10−04). The Tukey box plots represent the median and the 25th and 75th percentiles (interquartile range); points are displayed as outliers (represented by dots) if 1.5 times above or below the interquartile range (represented by whiskers). (C and D) For the same experiment as in A, the speed of advance of the leading edge (invasion speed through the indicated overlay; C; ***, P < 2.01 × 10−10) and the speed of individual cell migration (D; 100 cells tracked per conditions; ***, P < 3.49 × 10−23) were measured using manual tracking (see Materials and methods for details). n numbers for A to D: condition, movies analyzed; MCF10A LifeAct fibrillar collagen I, 14; MCF10A LifeAct GFR Matrigel, 19; DCIS.COM LifeAct fibrillar collagen I, 23; DCIS.COM LifeAct GFR Matrigel, 30. Statistical analysis: Student’s t test (unpaired, two tailed, unequal variance).

Filopodia dynamics can be analyzed using FiloQuant in circular invasion assays. (A and B) MCF10A and DCIS.COM cells stably expressing LifeAct-mRFP were plated in circular invasion assays and left to invade through fibrillar collagen I or GFR Matrigel for 3 d before being imaged live using an SDC microscope (100× objective, EMCCD camera) for over 9 h (one picture every 3 min). Filopodia were then automatically detected using the automated version of FiloQuant (software 3) and tracked using TrackMate (ImageJ-based tracking tool). For each condition, stills of single z planes with filopodia detected by FiloQuant (magenta) are displayed (bar, 25 µm) at different time points. In addition, time projections of detected filopodia as well as filopodia tracks are shown. The time projections were directly generated by FiloQuant and are color coded as a function of time. For filopodia tracking, tracking files, generated by FiloQuant, were entered into TrackMate for automated tracking. Filopodia tracks generated by TrackMate are color coded as a function of the track starting time (track index; A). For each condition, the percentage of stable filopodia (lifetime > 3 min) and the mean lifetime of stable filopodia were quantified from two independent experiments and displayed as Tukey box plots (B; ***, P < 2.79 × 10−04). The Tukey box plots represent the median and the 25th and 75th percentiles (interquartile range); points are displayed as outliers (represented by dots) if 1.5 times above or below the interquartile range (represented by whiskers). (C and D) For the same experiment as in A, the speed of advance of the leading edge (invasion speed through the indicated overlay; C; ***, P < 2.01 × 10−10) and the speed of individual cell migration (D; 100 cells tracked per conditions; ***, P < 3.49 × 10−23) were measured using manual tracking (see Materials and methods for details). n numbers for A to D: condition, movies analyzed; MCF10A LifeAct fibrillar collagen I, 14; MCF10A LifeAct GFR Matrigel, 19; DCIS.COM LifeAct fibrillar collagen I, 23; DCIS.COM LifeAct GFR Matrigel, 30. Statistical analysis: Student’s t test (unpaired, two tailed, unequal variance).

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