Label‐Free Cell‐Based Dynamic Mass Redistribution Assays

Hannah J. Gitschier1, Audrey B. Bergeron1, David H. Randle1

1 Corning Life Sciences, Kennebunk, Maine
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch130205
Online Posting Date:  March, 2014
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Abstract

Label‐free cell‐based assays offer a powerful approach to drug discovery and compound profiling for endogenously expressed receptors in a variety of cell types, including primary and stem cells. Dynamic mass redistribution (DMR) responses in whole cells following receptor stimulation provide phenotypic activity profiles that are readily amenable to evaluation of compound pharmacology. Protocols are provided in this unit to obtain DMR response profiles in adherent and suspension cells, and then to use known tool compounds to delineate the biology of the underlying signaling pathways from the information‐rich kinetic traces that are recorded. Curr. Protoc. Chem. Biol. 6:39‐51 © 2014 by John Wiley & Sons, Inc.

Keywords: cell surface receptors; phenotypic assays; drug discovery; signal transduction pathways; label‐free; optical biosensor; dynamic mass redistribution; primary cells

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Agonist DMR Responses in Adherent Tumor Cell Lines
  • Alternate Protocol 1: Agonist DMR Responses in Primary Cells
  • Basic Protocol 2: Dual‐ or Triple‐Addition Assays for Antagonist and Allosteric Modulation DMR Responses
  • Alternate Protocol 2: Dual‐Addition DMR Assays for Signaling Pathway Deconvolution
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Agonist DMR Responses in Adherent Tumor Cell Lines

  Materials
  • Cell lines of interest, e.g.:
    • A431 cells (ATCC #CRL‐1555)
    • A549 cells (ATCC #CCL‐185)
    • HEK‐293 cells (ATCC #CRL‐1573)
    • CHO‐K1 cells (ATCC #CCL‐61)
    • HeLa cells (ATCC #CCL‐2)
    • PC‐3 cells (ATCC #CRL‐1435)
  • Appropriate complete cell culture medium (e.g., DMEM, F‐12, RPMI, IMDM; see annotation to step 2) containing fetal bovine serum (FBS)
  • Assay buffer (see recipe; also see annotation to step 8)
  • Test compounds
  • Appropriate positive control agonist [e.g., TRAP‐5 amide (SFLLR), histamine, isoproterenol, nicotinic acid, EGF, ATP]
  • 15‐ml conical centrifuge tubes (e.g., Falcon)
  • Centrifuge
  • 384‐well microplate with embedded optical biosensors (test plate; e.g., Corning, cat. no. 5040)
  • 384‐well polypropylene storage microplate (source plate)
  • Label‐free microplate reader (e.g., Corning EPIC BT System, cat. no. 5053)
  • Data analysis software (e.g., Corning EPIC Analyzer, Microsoft Excel, or GraphPad Prism)

Alternate Protocol 1: Agonist DMR Responses in Primary Cells

  Additional Materials (also see protocol 1)
  • Primary cells of interest, e.g.:
    • GIBCO human neural stem cells (hNSCs; Invitrogen, cat. no. N7800‐100)
    • iCell cardiomyocytes (Cellular Dynamics, cat. no. CMC‐100‐110‐001)
    • Human aortic endothelial cells (Cascade Biologics, cat. no. C‐006‐5C)
    • Human umbilical vein endothelial cells (Corning, cat. no. 354151)
    • Peripheral blood mononuclear cells (AllCells, cat. no. PB006F)
    • Appropriate cell culture/differentiation medium with supplements, e.g.: IMDM, KnockOut DMEM/F‐12 with Stempro supplement, BSA, EDTA; Medium 200 with low‐serum growth supplement; iCell cardiomyocyte plating medium

Basic Protocol 2: Dual‐ or Triple‐Addition Assays for Antagonist and Allosteric Modulation DMR Responses

  Materials
  • Appropriate agonist [e.g., carbamoylcholine chloride (Carbachol), a G q‐coupled muscarinic agonist)]
  • Appropriate allosteric modulator (e.g., BQCA, a muscarinic allosteric potentiator)
  • Appropriate antagonist (e.g., atropine, a muscarinic antagonist, or Iressa, an EGFR inhibitor)
  • Additional reagents and equipment for cell seeding, and buffer exchange, and label‐free assay (Basic Protoocol 1)

Alternate Protocol 2: Dual‐Addition DMR Assays for Signaling Pathway Deconvolution

  Additional Materials ( )
  • Pertussis toxin (Tocris, cat. no. 3097)
  • Cholera toxin (Sigma‐Aldrich, cat. no. C9903)
  • G q‐inhibiting component (University of Bonn, Institute of Pharmacological Biology, no. QIC)
  • Appropriate kinase inhibitors (e.g., CGP 53753 or GW 5074)
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Figures

Videos

Literature Cited

Literature Cited
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