Fluorescence Fluctuation Spectroscopy to Study Protein Interaction at Cell Contacts
Published: May 31, 2023
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 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…
Disclosures
The authors have nothing to disclose.
Materials
| DMEM growth medium | PAN-Biotech | P04-01548 | |
| DPBS w/o: Ca2+ and Mg2+ | PAN-Biotech | P04-36500 | |
| DPBS w: Ca2+ and Mg2+ | PAN-Biotech | P04-35500 | |
| Trypsin EDTA | PAN-Biotech | P10-023100 | |
| TurboFect Transfection Reagent | Thermo Fisher Scientific | R0531 | |
| HEK 293T cells | DSMZ | ACC 635 | |
| Alexa Fluor 488 NHS Ester | Thermo Fisher Scientific | A20000 | |
| Rhodamine B | Sigma-Aldrich | 83689-1G | |
| Plasmid DNA | Addgene | NA | See Dunsing et. al., MBoC 2017, for a detailed description of all plasmids |
| 6-well plate | Starlab | CC7672-7506 | |
| 35-mm glass bottom dishes | CellVis | D35-14-1.5-N | |
| Zeiss LSM780 confocal | Carl Zeiss | NA | |
| Neubauer cell counting chamber | Marienfeld | 640110 |
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Cite This Article
Fluorescence Fluctuation Spectroscopy to Study Protein Interaction at Cell Contacts. J. Vis. Exp. (Pending Publication), e21399, doi: (2023).