Parallel high-resolution imaging of leukocyte chemotaxis under agarose with Rho-family GTPase biosensors

George R.R. Bell, Dean E. Natwick, Sean Collins

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Neutrophils are key early responders in the innate immune response that use chemotaxis, the directed migration along chemical gradients, to reach sites of infection or inflammation. This process requires integrating inputs from cell surface receptors with the cell’s polarity and motility signaling network, in which highly dynamic and interconnected signaling by Rho-family GTPases plays a central role. To understand this fundamentally important behavior, we describe a high-resolution, under-agarose chemotaxis assay for use with neutrophil-like cell lines (HL-60 or PLB-985) or with primary neutrophils. We also describe how to use optical uncaging of chemoattractants to stimulate cells in this assay. These techniques are compatible with epifluorescence, total internal reflection fluorescence (TIRF), and confocal microscopy. Additionally, we cover how to measure the activities of Rho-family GTPases in this context using Förster resonance energy transfer (FRET)-based biosensors. The specific experimental steps outlined in this chapter include how to (1) set up the under-agarose assay, (2) optically pattern chemoattractant gradients, (3) image cells, and (4) conduct basic image analysis for FRET biosensors.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages71-85
Number of pages15
DOIs
StatePublished - Jan 1 2018

Publication series

NameMethods in Molecular Biology
Volume1821
ISSN (Print)1064-3745

Keywords

  • Assay
  • Cell Signaling
  • Chemotaxis
  • FRET biosensors
  • Rho GTPases

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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  • Cite this

    Bell, G. R. R., Natwick, D. E., & Collins, S. (2018). Parallel high-resolution imaging of leukocyte chemotaxis under agarose with Rho-family GTPase biosensors. In Methods in Molecular Biology (pp. 71-85). (Methods in Molecular Biology; Vol. 1821). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-8612-5_6