Figure 4. Quantitative analysis of sphere formation under different culture conditions. The left panel shows phase (A) and fluorescent (B) photomicrographs of murine retinal progenitor cells (RPCs). Examples of object thresholding and quantification using image analysis software are shown (C-F), with white indicating selected objects and red indicating rejected objects. Specifically, a given microscopic field is thresholded to select all spheres (C), small spheres (D), middle-sized spheres (E), and large spheres (F; none present in this image). The scale bar is 400 µm. Right panel: RPCs were cultured in medium containing epidermal growth factor (EGF) or EGF + glial cell line-derived neurotrophic factor (GDNF) for 5 days. Number and cross-sectional area of spherical cellular aggregates (spheres) larger than 870 µm² are shown for each time point. Spheres meeting threshold criteria increased in number along the time course in both conditions. Significantly greater numbers and larger area of the spheres were found in the EGF+GDNF condition, compared to EGF at day 5, with the earlier trend seen at day 1 and day 3 not reaching statistical significance by the criterion used. Data represent the mean of six samples from same plating (*p<0.05). x-axis shows different medium conditions at day 1, 3, and 5; y-axis shows the number of spheres. Different shading within the histogram shows sphere area (µm²). Black is for small spheres, light gray for middle-sized, and dark gray for large. Standard deviation was used to generate the error bars, which reflect the sphere numbers.