Chemotaxis curve

Multi-well modified Boyden chambers can be obtained from Neuro Probe Inc. (Gaithersburg, MD). Model AP48 has been widely used for endothelial cell chemotaxis assays. The apparatus consists of top and bottom acrylic plates, a silicon gasket and assembly screws. The bottom plate has 48 wells, each with 25 pL final volume. These correspond to holes on the top plate, and form the upper wells when the chamber is assembled. The filter (polycarbonate, 25 x 80 mm) is placed between the top and bottom plates, and a gasket is placed over the filter to create the seal. The apparatus can be purchased with a selection of accessories, such as curved forceps, filter clamps, and wipers. These are required to process the filters after use. Filters can also be obtained from Nucleopore Inc. (Pleasanton, CA) and Costar (Cambridge, MA). 

This analysis was performed separately for Cy3-cAMP bound to the anterior pseudopods and the posterior tails of the Dictyostelium cells undergoing chemotaxis, revealing that Cy3-cAMP receptor complexes on anterior pseudopods dissociated about three times faster than those on posterior tails (Fig. 13). The dissociation curves were fitted to a sum of two exponential functions, indicating the presence of at least two receptors that adopt different kinetic states. Further characterization of this difference in the receptor states between anterior and posterior region suggests that the difference in the receptor states... 

Fig. 13a, b Single-molecule analysis of the binding kinetics of Cy3-cAMP on Dictyostelium cells undergoing chemotaxis a receptor occupancy of the cell under a gradient of Cy3-cAMP. The arrow represents the direction of the source of Cy3-cAMP. Time is given in seconds. Individual Cy3-cAMP spots and the cell contour are traced b the release curves of Cy3-cAMP spots bound to the anterior pseudopods or the posterior tails. The release curves of bound Cy3-cAMP were fitted to two exponentials. At anterior pseudopod, k. =1.1 s" (71%) and 0.39 s" (29%). At the posterior taU, k i=0.39 s (76%) and 0.16 s (24%). Chemical gradients in cAMP concentration maybe converted into the differences in the receptor states... 

---