Label‐Free Dynamic Mass Redistribution and Bio‐Impedance Methods for Drug Discovery

Manuel Grundmann1

1 Section Cellular, Molecular and Pharmacobiology, Institute for Pharmaceutical Biology, University of Bonn, Bonn
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 9.24
DOI:  10.1002/cpph.24
Online Posting Date:  June, 2017
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Label‐free biosensors are increasingly employed in drug discovery. Cell‐based biosensors provide valuable insights into the biological consequences of exposing cells and tissues to chemical agents and the underlying molecular mechanisms associated with these effects. Optical biosensors based on the detection of dynamic mass redistribution (DMR) and impedance biosensors using cellular dielectric spectroscopy (CDS) capture changes of the cytoskeleton of living cells in real time. Because signal transduction correlates with changes in cell morphology, DMR and CDS biosensors are exquisitely suited for recording integrated cell responses in an unbiased, yet pathway‐specific manner without the use of labels that may interfere with cell function. Described in this unit are several experimental approaches utilizing optical label‐free system capturing dynamic mass redistribution (DMR) in living cells (Epic System) and an impedance‐based CDS technology (CellKey). In addition, potential pitfalls associated with these assays and alternative approaches for overcoming such technical challenges are discussed. © 2017 by John Wiley & Sons, Inc.

Keywords: label‐free; biosensor; DMR; CDS; phenotypic assay

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Table of Contents

  • Introduction
  • Basic Protocol 1: Standard Dynamic Mass Redistribution Assay (DMR) Assay
  • Alternate Protocol 1: DMR Assay With Cell Suspensions
  • Alternate Protocol 2: Antagonist‐Mode DMR Assay
  • Basic Protocol 2: Standard Bio‐Impedance (CDS) Assay Protocol
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Standard Dynamic Mass Redistribution Assay (DMR) Assay

  • HEK293 cells expressing the FFA2 receptor (FFA2‐HEK; contact Evi Kostenis at kostenis@uni‐ for cell line requests) growing in T‐75 (75‐cm2) flasks
  • Complete growth medium (see recipe)
  • Phosphate‐buffered saline (PBS; see recipe)
  • 0.05% (w/v) trypsin/0.02% (w/v) EDTA (e.g., Gibco, Thermo Fisher Scientific)
  • Assay buffer (see recipe)
  • 1 M propionic acid stock solution, aqueous (e.g., Sigma‐Aldrich)
  • Controls (see annotation to step 14, below)
  • 384‐well, fibronectin‐coated biosensor plate (e.g., Corning Cell Assay Plate 5042)
  • Multichannel and/or repeat pipettors
  • Multichannel manifold attached to cell culture vacuum pump (e.g., from Socorex, see Fig.  ; alternatively, use a liquid handling station that can be used for washing protocols, e.g., PerkinElmer JANUS automated workstation)
  • Centrifuge
  • DMR reader (e.g., Corning Epic BT reader or PerkinElmer EnSight)
  • 384‐well storage plates (e.g., Corning Costar 3657)
  • Liquid‐handling station capable of transferring all 384‐well formats at the same time (e.g., CyBi‐SELMA, Analytik Jena)
  • Incubator for label‐free readers without an on‐board temperature control unit (e.g., from Memmert)
  • PC workstation for processing label‐free data running e.g., EpicImager (Corning) or Kaleido Data Acquisition and Analysis Software (PerkinElmer), Microsoft Excel, and GraphPad Prism
  • Additional reagents and equipment for cell culture including trypsinization and cell counting (Phelan & May, 2016)

Alternate Protocol 1: DMR Assay With Cell Suspensions

  Additional Materials (also see protocol 1)
  • At least two ligands (antagonist, e.g. CATPB or GLPG0974, and an established agonist, e.g., propionic acid, acetic acid, 4‐CMTB)
  • Two 384‐well storage plates (e.g., Corning Costar 3657)

Alternate Protocol 2: Antagonist‐Mode DMR Assay

  • 0.1 mg/ml poly‐D‐lysine (PDL)
  • Phosphate‐buffered saline (PBS; see recipe)
  • HEK293 cells expressing the FFA2 receptor (FFA2‐HEK; see Grundmann 2016) growing in T‐75 (75‐cm2) flasks
  • 0.05% (w/v) trypsin/0.02% (w/v) EDTA (e.g., Gibco, Thermo Fisher Scientific)
  • Complete growth medium (see recipe)
  • Assay buffer (see recipe)
  • 1 M propionic acid stock solution, aqueous (e.g., Sigma‐Aldrich)
  • Controls (see annotation to step 15, below)
  • 384‐well cellular dielectric spectroscopy biosensor plate (e.g., CellKey Assay Plate, Molecular Devices)
  • Multichannel and/or repeat pipettors
  • Multichannel manifold attached to cell culture vacuum pump (e.g., from Socorex, see Fig.  ; alternatively, use a liquid handling station that can be used for washing protocols, e.g., PerkinElmer JANUS automated workstation)
  • Centrifuge
  • 384‐well storage plate (e.g., Corning Costar 3657)
  • Bio‐impedance CDS reader (e.g., Molecular Devices CellKey System)
  • PC workstation to process label‐free data (e.g., Molecular Devices CellKey 384 Software, Microsoft Excel, GraphPad Prism)
  • Additional reagents and equipment for cell culture including trypsinization and cell counting (Phelan & May, 2016)
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Literature Cited

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