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and wing cells (Figure 1 B2 & C2). The field of view counted for the inverted scope images was only a 8.8 x 6.7 inch (in photoshop with resolution of 72 pixels/inch) portion of the whole drop. The same size area was counted in each instance.

Treatment with two concentrations of poly-l-lysine proved the most successful in our experiment. As visualized under the bright field microscope after Alcian Blue staining, the 0.1mg/ml and 0.5mg/ml concentration of poly-l-lysine stimulated chondrogenesis in both the wing and hind limb (Figure 2 B3, B4, C3, & C4). It didn't seem to make a difference what concentration we used. Both showed similar results. However, under the inverted scope, the cell counts in the specified area were similar to the RA-treated cell counts. These face poly-l- lysine counts were also very similar to the same counts for wing and hind limb cells treated with poly-l-lysine (Figure 1 A3 & A4).

Additionally, we attempted to treat all wells with guanidine HCl in order to remove the Alcian Blue stain from the cells, as suggested in Ting-Xin et al. (1992). The guanidine HCl did not work in this manner. Therefore, we were not able to measure the amount of dye absorbed by the nodules through use of a spectrophotometer.

Given the time constraint, it was not possible to pinpoint the cause and correct the problem of facial cell adherence. For future experimentation, several possible solutions to this problem present themselves. Since, fibronectin adheres to the ECM and allows the cells to adhere to the well, one solution may be to coat the bottom of the wells with fibronectin prior to adding the sample of facial cells. Additionally, previous experiments have shown that wing and hind limb buds react differently to different serums (Downie & Newman 1994&1995). As the crainofacial cells may also react differently, one approach to promoting cell adherence would be to culture face cells in several different types of serum to determine which is most effective.

©Cebra-Thomas, 2000

Last Modified: 2 May 2000

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