Visualizing in vitro trafficking

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Here we present a detailed guide for performing in vitro trafficking assays. These are high-resolution light microscopy assays designed to look at the cytoskeletal filament-based trafficking of cellular organelles. The strategy is to partially purify organelles from lysed mammalian cells and freeze them as single-use aliquots. The organelles are then thawed and allowed to bind microtubule and actin filaments that have been coated onto handmade optical microchambers. Time lapse multichannel fluorescence microscopy is then performed to identify specific vesicles and associated proteins and to observe and quantify how the material is transported. These protocols were initially developed to study rodent liver endosomes but are adapted here for the study of cultured cells and include commentary on their use with other types of organelles.

Original languageEnglish (US)
Title of host publicationThe Cytoskeleton
Subtitle of host publicationImaging, Isolation, and Interaction
Pages19-39
Number of pages21
DOIs
Publication statusPublished - Apr 2 2013

Publication series

NameNeuromethods
Volume79
ISSN (Print)0893-2336
ISSN (Electronic)1940-6045

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Keywords

  • Actin
  • Cellular organelles
  • Endosomes
  • In vitro trafficking
  • Microtubules
  • Time lapse imaging

ASJC Scopus subject areas

  • Neuroscience(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Pharmacology, Toxicology and Pharmaceutics(all)
  • Psychiatry and Mental health

Cite this

Murray, J. W. (2013). Visualizing in vitro trafficking. In The Cytoskeleton: Imaging, Isolation, and Interaction (pp. 19-39). (Neuromethods; Vol. 79). https://doi.org/10.1007/978-1-62703-266-7-2