Cell Aggregation Assay to Detect Trans Interactions between Cell Adhesion 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.