Fig. 2

Formation of murine SVF-derived spheroids in low adherence plates and hanging drop cultures. A Serial dilutions of 750,000, 375,000, and 187,500 SVF cells/ml (from upper to bottom row) were seeded in low attachment plates (LA, left panels) or hanging drops (right panels), as indicated, and spheroids formation was checked after 24 and 48 h. Only in low attachment plate experiments, cultures were mechanically disaggregated after 24 h, diluted 1:5 and re-plated for additional 24-h incubation. All images were taken using a bright-field microscope with 10 × magnification. 100 µm scale bar size. B Spheroids formed in low attachment plates or hanging drop were photographed (bright field microscopy) and subjected to particle analysis with ImageJ software. The number of analyzed spheroids was: 1) hanging drop: 187,500 cells/ml N = 235; 375,000 cells/ml N = 234; 750,000 cells/drop N = 475; 2) low attachment plate: 187,500 cells/ml N = 37; 375,000 cells/ml N = 48; 750,000 cells/ml N = 404. Size distribution by calculation of feret expressed as mean and standard deviation of each population. Welch-ANOVA; W (5, 224) = 129.9; p < 0.0001; Games–Howell post-test. C Yield of spheroid formation per formation unit with 4 runs per group. Two-way ANOVA, Droplet vs LA Plate: F (1, 18) = 266.7; P < 0.0001; Initial cell concentration: F (2, 18) = 19.74; P < 0.0001; Tukey's post-test. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns not significant