This study investigates transcellular interactions between synaptic cell adhesion molecules in human embryonic kidney cells. Using a cell aggregation assay, the research aims to visualize these interactions through fluorescence microscopy.
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.
48 hours after transfecting the HEK293T cells, harvest the cells from the 6-well plate for aggregation. First, wash each well twice with PBS. Next, to gently disassociate the cells, add 1 milliliter of 10-millimolar EDTA to each well. Then, incubate the plate at 37 degrees Celsius.
After five minutes of incubation, gently tap the plate to detach the cells, and harvest each well into a separate 15-milliliter conical tube. Centrifuge the tubes at 500 x g at room temperature for 5 minutes.
While the cells are pelleting, prepare six incubation tubes by labeling the tops of microcentrifuge tubes with the experimental conditions. Every permutation of GFP and mCherry should be used.
Remove the supernatants from the 15-milliliter conical tubes, and resuspend the cells in 500 microliters of medium. Using a hemocytometer, count the cells in each conical tube. Then, aliquot 200,000 cells from each condition into the appropriate incubation tube for a one-to-one mix in a total volume of 500 microliters. Place the incubation tubes in a slow tube rotator at room temperature.
To assess aggregation, at baseline, and again after 60 minutes, pipette 40 microliters from the incubation tube onto a charged microscope slide. Baseline acquisition should be done as quickly as possible after the addition of cells to the microcentrifuge tube.
Image the slide under fluorescence in both the green and red channels. For each slide, capture images of three different fields of view in one focal plane.
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