Figure 7.
Figure 7. Analysis of filopodia density in 3D tumor spheroids using FiloQuant. (A) MCF10A, MCF10AT, and DCIS.COM cells were seeded as single cells on GFR Matrigel and left to form spheroids over 7 or 14 d, fixed, stained, and imaged using a confocal microscope. For each condition, representative single z planes (see Fig. S4 A for cartoon depicting imaging plane) as well as magnified areas (red squares) are displayed. Bars, 25 µm. (B) DCIS.COM cells were seeded as in A and left to form spheroids for 21 d, fixed, stained and imaged using a confocal microscope. A representative single z plane, magnified region (red square) and an Imaris-generated 3D reconstruction are displayed. Bars, 25 µm. (C) MCF10A and DCIS.COM cells were seeded as in A and allowed to form spheroids for 5 d, fixed, stained, and imaged using an SDC microscope (100× objective, CMOS camera). Representative single z planes and magnified areas (red square) are displayed. Bars: (main) 20 µm; (inset), 5 µm. Filopodia density at spheroid borders was quantified using FiloQuant. Results from two independent experiments are displayed as Tukey box plots (condition, fields of view analyzed: MCF10A, 18; DCIS.COM, 11; ***, P = 3.06 × 10−05). 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). The filopodia detection images generated by FiloQuant are displayed in Fig. S4. (D) DCIS.COM LifeAct cells were seeded as in A and allowed to form spheroids for 3 d before being imaged live using an SDC microscope (100× objective, EMCCD camera) for over 3 h (one picture every 2 min). Filopodia were then automatically detected using FiloQuant and tracked using TrackMate as in Fig. 6 B. Stills of single z planes with filopodia detected by FiloQuant (magenta) and a time projection of the detected filopodia and filopodia tracks are displayed. Bar, 25 µm. The time projections are color coded as a function of time. Filopodia tracks are color coded as a function of the track length to highlight filopodia stability.

Analysis of filopodia density in 3D tumor spheroids using FiloQuant. (A) MCF10A, MCF10AT, and DCIS.COM cells were seeded as single cells on GFR Matrigel and left to form spheroids over 7 or 14 d, fixed, stained, and imaged using a confocal microscope. For each condition, representative single z planes (see Fig. S4 A for cartoon depicting imaging plane) as well as magnified areas (red squares) are displayed. Bars, 25 µm. (B) DCIS.COM cells were seeded as in A and left to form spheroids for 21 d, fixed, stained and imaged using a confocal microscope. A representative single z plane, magnified region (red square) and an Imaris-generated 3D reconstruction are displayed. Bars, 25 µm. (C) MCF10A and DCIS.COM cells were seeded as in A and allowed to form spheroids for 5 d, fixed, stained, and imaged using an SDC microscope (100× objective, CMOS camera). Representative single z planes and magnified areas (red square) are displayed. Bars: (main) 20 µm; (inset), 5 µm. Filopodia density at spheroid borders was quantified using FiloQuant. Results from two independent experiments are displayed as Tukey box plots (condition, fields of view analyzed: MCF10A, 18; DCIS.COM, 11; ***, P = 3.06 × 10−05). 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). The filopodia detection images generated by FiloQuant are displayed in Fig. S4. (D) DCIS.COM LifeAct cells were seeded as in A and allowed to form spheroids for 3 d before being imaged live using an SDC microscope (100× objective, EMCCD camera) for over 3 h (one picture every 2 min). Filopodia were then automatically detected using FiloQuant and tracked using TrackMate as in Fig. 6 B. Stills of single z planes with filopodia detected by FiloQuant (magenta) and a time projection of the detected filopodia and filopodia tracks are displayed. Bar, 25 µm. The time projections are color coded as a function of time. Filopodia tracks are color coded as a function of the track length to highlight filopodia stability.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal