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Rho GTPases pp 71-85 | Cite as

Parallel High-Resolution Imaging of Leukocyte Chemotaxis Under Agarose with Rho-Family GTPase Biosensors

  • George R. R. Bell
  • Dean E. Natwick
  • Sean R. CollinsEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1821)

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.

Key words

Chemotaxis Assay Cell Signaling FRET biosensors Rho GTPases 

Notes

Acknowledgments

We would like to thank the following funding sources for their support of this work. SRC thanks the Sidney Kimmel Foundation for the 2017 Kimmel Scholar Award, and the NIH for the Director’s New Innovator Award DP2-HD-094656. DEN thanks the NIH T32 GM007377 training grant, while GRRB thanks the National Science Foundation Graduate Research Fellowship (Grant Number 1650042) for support.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • George R. R. Bell
    • 1
  • Dean E. Natwick
    • 1
  • Sean R. Collins
    • 1
    Email author
  1. 1.Department of Microbiology and Molecular GeneticsUniversity of California, DavisDavisUSA

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