Fluorescence Fluctuation Spectroscopy to Study Protein Interaction at Cell Contacts

In This Article

Abstract

Source: Dunsing, V. et al. A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts. J. Vis. Exp. (2018).

This video demonstrates the use of fluorescence fluctuation spectroscopy to detect the interaction among cell surface proteins at cell-cell contacts. By expressing the transmembrane adhesion receptor of interest labeled with a fluorescent protein and mixing two different cell populations harboring two spectrally separated fluorescent labels, the trans-interaction between the receptors of two neighboring cells with different-colored fluorescence is assessed via cross-correlation in the fluctuations of fluorescence intensity.

Protocol

1. Sample Preparation: Cell-Cell Mixing Assay

NOTE: The following protocol describes the mixing procedure for adherent cells. It may be modified for cells cultured in suspension.

Seed an appropriate number of cells on a 6-well plate, e.g., 800,000 HEK 293T cells (counted with a Neubauer counting chamber), a day before transfection. The number can be modified depending on the time between seeding and transfection and adjusted for other cell types. To perform a basic experiment (i.e., proteins of interest and negative control), prepare at least 4 wells. Culture cells at 37 °C, 5% CO₂

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Results

Fluorescence correlation spectroscopy diagram shows APLP1 protein interactions and pixel analysis data.
Figure 1. Experimental workflow and schematic representation of scanning fluorescence cross-correlation spectroscopy and cross-correlation number and brightness analysis at cell-cell contacts. (A) Scheme of sample preparation: Two ce.......