Fig. 3

Time course for aggregation and maturation of spheroids. a Dispersed WT-GFP plus unlabeled E-cadherin-deficient ES cells were mixed and allowed to aggregate in suspension culture. Representative images of GFP signals and overlayed on bright field were shown for 0, 12, 24, and 48-h following mixing. b Images of 24-h spheroids from WT-GFP plus unlabeled wild-type or E-cadherin-deficient ES cells are shown. Optical sectioning by confocal microscopy of representative individual spheroid was also shown. c Images from 48-h spheroids are shown. Scale bar denotes 100 μm. The Z-stack confocal sectionings of the aggregates are included as supplementary results (Supplemental movie 3, 4, 5, 6). d An example shows the approach to quantitate the distribution of cell types in an aggregate from WT-GFP plus E-cadherin (−/−) ES cells. The image of the aggregate was divided into equal areas of outer ring or inner circle, defined as the region of interest (ROI). The intensities of GFP or DAPI in the ROI were quantitated by Image J program. e The distribution of GFP signal in the outer ROI (region of interest) was calculated and averaged from multiple aggregates, with standard errors indicated. The numbers of aggregates analyzed were 59 and 228 (wildtype), and 39 and 121 (wildtype plus E-cadherin null), for 24- and 48-h timepoint respectively. The differences are statistically significant by Student’s t-test, indicated by “*” and “**” for a p value < 0.005 in both cases. f The distribution of DAPI signal was shown as controls and for comparison