Fluorescence Fluctuation Spectroscopy to Study Protein Interaction at Cell Contacts

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.