A Unique High‐Throughput Assay to Identify Novel Small Molecule Inhibitors of Chemotaxis and Migration

Xin‐Hua Liao1, Alan R. Kimmel2

1 Fujian Key Laboratory for Translational Research in Cancer and Neurodegenerative Diseases, Institute for Translational Medicine, and Key Laboratory of Ministry of Education for Gastrointestinal Cancer, Fujian Medical University, Fuzhou, Fujian, 2 Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 12.11
DOI:  10.1002/cpcb.17
Online Posting Date:  March, 2017
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Chemotaxis and cell migration play pivotal roles in normal physiological processes such as embryogenesis, inflammation, and wound healing, as well as in pathological processes including chronic inflammatory disease and cancer metastasis. Novel chemotaxis/migration inhibitors are desirable for developing effective therapeutics and probing molecular mechanisms. We describe a fluorescence‐based phenotypic assay in a 1536‐well plate format for high‐throughput screening of novel inhibitors of chemotaxis/migration within complex libraries of thousands of compounds. Although the assay utilizes the unique cellular response properties of Dictyostelium, the compounds identified are able to inhibit chemotaxis of mammalian cells. In addition, a parallel cell cytotoxicity counter‐screen with an ATP content assay is described that eliminates cytotoxic compounds from the screen. This novel compound screening approach enables rapid identification of novel lead compounds that inhibit chemotaxis in human and other cells for drug development and research tools. © 2017 by John Wiley & Sons, Inc.

Keywords: cell migration; chemotaxis; LOPAC; acumen eX3; Dictyostelium

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Table of Contents

  • Introduction
  • Basic Protocol 1: Miniaturized Dictyostelium Aggregation Assay for Screening of Chemotaxis/Migration Inhibitors
  • Basic Protocol 2: Dictyostelium Cytotoxicity Assay for Eliminating Cytotoxic Compounds
  • Support Protocol 1: Data Analysis
  • Support Protocol 2: Re‐Confirmation and Validation of Chemotaxis Inhibition
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Miniaturized Dictyostelium Aggregation Assay for Screening of Chemotaxis/Migration Inhibitors

  • Dictyostelium strain [cotB]:GFP (dictyBase, strain i.d. DBS0236466) (Basu et al., )
  • D3‐T medium (see recipe)
  • DB starvation buffer (see recipe)
  • DMSO (Sigma‐Aldrich, cat. no. D2650)
  • Library of Pharmacologically Active Compounds: LOPAC1280 (Sigma‐Aldrich, #LO1280) or other compound collections
  • Control compound: latrunculin A (Sigma‐Aldrich, cat. no. L5163)
  • 25‐cm² canted‐neck cell culture flask, sterile
  • Incubator with temperature controlled at 21°C
  • 250‐ml or larger glass or plastic Erlenmeyer flask, sterile
  • Orbital shaker at 21°C
  • Centrifuge 5810R (Eppendorf)
  • Evolution P3 system (PerkinElmer)
  • 384‐well plates
  • 1536‐well clear‐bottom plates (Aurora Biotechnologies)
  • Multidrop Combi Reagent Dispenser (Thermo Fisher Scientific)
  • Kalypsys microplate metal lids
  • Peelable aluminum microplate sealers (Agilent Technologies, cat. no. 24210‐001)
  • Agilent PlateLoc thermal microplate sealer
  • Pintool Station (Kalypsys)
  • Acumen eX3 (TTP LabTech)
  • Additional reagents and equipment for counting cells (unit 1.1; Phelan, )

Basic Protocol 2: Dictyostelium Cytotoxicity Assay for Eliminating Cytotoxic Compounds

  • Hygromycin B (Sigma‐Aldrich)
  • ATPlite assay kit (PerkinElmer, cat. no. 6016731)
  • CCD‐imager based ViewLux plate reader (PerkinElmer)
  • Additional reagents and equipment for Dictyostelium aggregation assay ( protocol 1)
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Literature Cited

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