Cell Aggregation Assay to Detect Trans Interactions between Cell Adhesion Molecules
Transcript
In the nervous system, transcellular interactions — interactions between specific synaptic cell adhesion molecules, transmembrane proteins, on presynaptic and postsynaptic neurons — form a complex at synapses, mediating trans-synaptic signaling.
To detect transcellular interactions between synaptic cell adhesion molecules expressed on human embryonic kidney cells via a cell aggregation assay, begin with transfected cell suspensions.
One transfected cell population expresses the desired presynaptic cell adhesion molecule and green fluorescent protein; the other expresses the specific postsynaptic cell adhesion molecule and red fluorescent protein. Two cell populations expressing green and red fluorescent proteins are also obtained.
Centrifuge the cells. Resuspend the cells in suitable media supplemented with calcium ions. Mix both populations' transfected cells equally in a tube. Repeat for the other cell populations.
During incubation, the calcium ions bind to a specific site on the presynaptic protein expressed in a cell population. This increases the presynaptic protein's interaction with the other cell population's postsynaptic ligand, facilitating binding, mediating heterophilic cell adhesion between these cells, and leading to cell aggregation.
Under a fluorescence microscope, image the cells using suitable channels, visualizing the green and red fluorescence of the cells. Process the images.
No aggregation is seen in the cell populations expressing only the fluorescent proteins. Cell aggregation is visualized as yellow due to overlap between the green and red fluorescent cells expressing the synaptic cell adhesion molecules, suggesting transcellular interactions between the molecules.
