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Fluorescence Fluctuation Spectroscopy to Study Protein Interaction at Cell Contacts
Overview
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 perfor.......
Representative Results
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 cell populations transfected with the protein of interest (e.g., APLP1) fused to two spectrally distinct fluorescent proteins (e.g., mEYFP and mCardina.......
This article has been published
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Source: Dunsing, V. et al. A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts. J. Vis. Exp. (2018).
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