Fluorescence Fluctuation Spectroscopy to Study Protein Interaction at Cell Contacts

Cell-cell interactions — mediated by proteins at neighboring cell surfaces — can be studied in live cells using a fluorescence fluctuation spectroscopy assay.

First, take separate cell cultures expressing an adhesion receptor — a transmembrane protein. The receptor in the cells from the two populations is labeled with spectrally-separated fluorescent proteins. These fluorescent labels are present on the receptor's cytoplasmic region, preventing interference with the cell-cell interaction.

Discard the medium and add trypsin, facilitating cell detachment. Mix the two cell populations and incubate, allowing the neighboring cells' receptors to form homotypic trans-interactions.

Place the cells under a confocal microscope. Illuminate the sample using laser sources of appropriate wavelengths, causing emission from both fluorescent labels. Locate two neighboring cells with different-colored fluorescence signals in contact.

Acquire a line scan — perpendicular to the region of cell membranes in contact — using the spectral channel of both fluorescent labels. The laser focuses on a small volume of the sample — the detection volume. As the fluorescent-labeled receptors diffuse in and out of t