Live Imaging of ESCRT Proteins in Microfluidically Isolated Hippocampal Axons

Methods Mol Biol. 2019:1998:117-128. doi: 10.1007/978-1-4939-9492-2_9.

Abstract

Live imaging of microfluidically isolated axons permits study of the dynamic behavior of fluorescently tagged proteins and vesicles in these neuronal processes. We use this technique to study the motility and transport of ESCRT proteins in axons of primary hippocampal neurons. This chapter details the preparation of microfluidic chambers, as well as the seeding, fluidic isolation, and lentiviral transduction of hippocampal neurons in these chambers, optimized for the study of ESCRT protein dynamics.

Keywords: Axonal isolation; Axonal transport; ESCRT; Hippocampal neuron; Live cell imaging; Microfluidic; Primary neuronal culture.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Axonal Transport
  • Axons / metabolism*
  • Cells, Cultured
  • Endosomal Sorting Complexes Required for Transport / genetics
  • Endosomal Sorting Complexes Required for Transport / metabolism*
  • Fluorescent Dyes / chemistry
  • Genetic Vectors
  • HEK293 Cells
  • Hippocampus / cytology
  • Humans
  • Intravital Microscopy / methods*
  • Lentivirus / genetics
  • Microfluidic Analytical Techniques / instrumentation
  • Microfluidic Analytical Techniques / methods*
  • Molecular Imaging / methods*
  • Molecular Probes / chemistry
  • Molecular Probes / genetics
  • Primary Cell Culture / methods
  • Rats
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Transfection

Substances

  • Endosomal Sorting Complexes Required for Transport
  • Fluorescent Dyes
  • Molecular Probes
  • Recombinant Proteins