This article describes a fluorescence fluctuation spectroscopy assay to study cell-cell interactions in live cells. The method involves using fluorescently labeled adhesion receptors to observe interactions between neighboring cells.
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 the detection volume, the fluorescence intensity fluctuates.
If the receptors of the neighboring cells do not interact, they move individually, leading to independent fluctuations in their fluorescence intensity with a low cross-correlation.
If the neighboring cells in contact interact via their receptors, the interacting proteins diffuse together, causing a correlated fluctuation in their fluorescence intensity.
Transfer the cell solution of one well to the corresponding well. Mix gently by pipetting a few times up and down, then, seed the mixed cells on a 35-milliliter glass-bottomed dish, and culture the seeded cells at 37 degrees Celsius and 5% carbon dioxide for one day.
In the laser-scanning confocal microscope software, set up the optical path. To avoid spectral cross-talk, select two separate tracks to excite and detect mEGFP or mEYFP and mCherry or mCardinal sequentially, and select "Switch Tracks Every Line." For the detection, use appropriate filters for both channels.
Place the dish containing the mixed cells on the sample holder. After waiting 10 minutes to ensure temperature equilibration and to reduce focus drift, focus on the cells using the Transmission Light in the "Locate" menu. Search for a pair of 'red' and 'green' cells in contact with each other.
Next, select a scan path perpendicular to cell-cell contact using the "Crop" button. "Zoom" to achieve a pixel size of 50 to 200 nanometers and select "Line" in "Scan Mode." Set "Frame Size" to 128 by 1 pixels. Set "Scan Speed" to the maximum allowed value, then set cycles to 100,000 to 500,000.
Following this, choose the appropriate laser powers. Set the detectors to "Photon Counting" Mode. Press "Start Experiment" to start the acquisition.
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