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. Author manuscript; available in PMC: 2010 May 26.
Published in final edited form as: FASEB J. 2005 May 18;19(10):1371–1373. doi: 10.1096/fj.04-2770fje

Figure 3.

Figure 3

Spheroid formation by stromal cells. Primary keratocytes in mitogen-free medium (A) attach and spread with a dendritic morphology. After 1 wk in medium containing 10 ng/ml FGF and ITS (B), cells form refractile aggregates. After 3 wk (C), aggregates become spherical and could be readily separated from individual cells (D). Live cells in spheres were stained with calcein AM (green) and propidium iodide (red) to show live and dead cells (E). Fixed spheres (F) were stained for keratan sulfate (green) and keratocan (red). Photos E and F are optical sections through the center of a sphere using confocal microscopy as described in Materials and Methods. RNA from spheres and from cells not aggregated into spheres from the same culture was subjected to analysis by quantitative RT-PCR for keratocan mRNA as described in Materials and Methods. mRNA pools were compared with those of noncultured stromal cells and with corneal fibroblasts in G. Bars in A–D = 100 μm and in E–F = 50 μm.