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Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells.

Authors
  • Mailfert, Sebastien1
  • Wojtowicz, Karolina2
  • Brustlein, Sophie1
  • Blaszczak, Ewa3
  • Bertaux, Nicolas4
  • Łukaszewicz, Marcin5
  • Marguet, Didier6
  • Trombik, Tomasz7
  • 1 CNRS, Inserm, Centre d'Immunologie de Marseille-Luminy, Aix Marseille Univ.
  • 2 Faculty of Biotechnology, University of Wroclaw.
  • 3 Faculty of Biological Sciences, University of Wroclaw.
  • 4 CNRS, Centrale Marseille, Institut Fresnel, Aix Marseille Univ.
  • 5 Faculty of Biotechnology, University of Wroclaw; [email protected]
  • 6 CNRS, Inserm, Centre d'Immunologie de Marseille-Luminy, Aix Marseille Univ; [email protected]
  • 7 Faculty of Biotechnology, University of Wroclaw; [email protected]
Type
Published Article
Journal
Journal of Visualized Experiments
Publisher
MyJoVE Corporation
Publication Date
Nov 12, 2020
Issue
165
Identifiers
DOI: 10.3791/61823
PMID: 33252108
Source
Medline
Language
English
License
Unknown

Abstract

Dynamic biological processes in living cells, including those associated with plasma membrane organization, occur on various spatial and temporal scales, ranging from nanometers to micrometers and microseconds to minutes, respectively. Such a broad range of biological processes challenges conventional microscopy approaches. Here, we detail the procedure for implementing spot variation Fluorescence Correlation Spectroscopy (svFCS) measurements using a classical fluorescence microscope that has been customized. The protocol includes a specific performance check of the svFCS setup and the guidelines for molecular diffusion measurements by svFCS on the plasma membrane of living cells under physiological conditions. Additionally, we provide a procedure for disrupting plasma membrane raft nanodomains by cholesterol oxidase treatment and demonstrate how these changes in the lateral organization of the plasma membrane might be revealed by svFCS analysis. In conclusion, this fluorescence-based method can provide unprecedented details on the lateral organization of the plasma membrane with the appropriate spatial and temporal resolution.

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