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Applying label-free dynamic mass redistribution technology to frame signaling of G protein–coupled receptors noninvasively in living cells

Nature Protocols volume 6, pages 1748–1760 (2011)Cite this article

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Abstract

Label-free dynamic mass redistribution (DMR) is a cutting-edge assay technology that enables real-time detection of integrated cellular responses in living cells. It relies on detection of refractive index alterations on biosensor-coated microplates that originate from stimulus-induced changes in the total biomass proximal to the sensor surface. Here we describe a detailed protocol to apply DMR technology to frame functional behavior of G protein–coupled receptors that are traditionally examined with end point assays on the basis of detection of individual second messengers, such as cAMP, Ca2+ or inositol phosphates. The method can be readily adapted across diverse cellular backgrounds (adherent or suspension), including primary human cells. Real-time recordings can be performed in 384-well microtiter plates and be completed in 2 h, or they can be extended to several hours depending on the biological question to be addressed. The entire procedure, including cell harvesting and DMR detection, takes 1–2 d.

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Figure 1: Principle of DMR detection.
Figure 2
Figure 3: Gαi/o GPCR activity can be assayed directly in primary human neutrophils.
Figure 4: The FFA1 receptor has an identical signaling profile and pharmacology in both adherent and suspension cells.
Figure 5: Determination of the dynamic nature of agonist and antagonist action in DMR assays.
Figure 6: Dynamic mass redistribution analysis of three prototypical GPCRs in recombinant CHO cells and deduction of concentration-effect curves from DMR signals.
Figure 7: Activity fingerprints of the adenylyl cyclase activator forskolin (Fsk) in a range of different cellular backgrounds.
Figure 8: Activity profiles of a selective small-molecule FFA1 agonist are not consistent across diverse cellular backgrounds.
Figure 9: Signaling profiles for a given cell type do not necessarily represent signaling pathway indicators.
Figure 10: Selected DMR experiments that required troubleshooting to obtain meaningful results.

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Acknowledgements

We thank U. Rick, M. Vasmer-Ehses and T. Kögler for expert technical assistance and Corning for providing us with the Epic system. This work was supported by the DFG (Deutsche Forschungsgemeinschaft, German Research Foundation) grants KO 1582/3-1 to E.K. and MO 821/2-1 to K.M. D.K. is a member of the DFG-funded Research Training Group GRK 677, W.S. is a member of the NRW International Graduate Research School Biotech Pharma. We thank M. De Amici and U. Holzgrabe (University of Milan, Italy, and University of Würzburg, Germany) for kindly providing Hybrid 1 (ref. 15), T. Ulven (University of Southern Denmark, Denmark) for TM30089 (ref. 31) and TUG424 (ref. 30), and Astellas Pharma (Osaka, Japan) for providing us with YM-254890 (ref. 32).

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Authors and Affiliations

  1. Molecular-, Cellular- and Pharmacobiology Section, Institute of Pharmaceutical Biology, University of Bonn, Bonn, Germany

    Ralf Schröder, Johannes Schmidt, Stefanie Blättermann, Lucas Peters, Manuel Grundmann, Nicole Merten, Christel Drewke, Jesus Gomeza & Evi Kostenis

  2. Pharmacology and Toxicology Section, Institute of Pharmacy, University of Bonn, Bonn, Germany

    Nicole Janssen, Wiebke Seemann, Dorina Kaufel & Klaus Mohr

  3. Molecular Pharmacology Group, Institute of Neuroscience and Psychology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Scotland, UK

    Graeme Milligan

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Contributions

R.S., J.S., S.B., L.P., N.J., M.G., W.S., D.K. and N.M. designed and performed the experiments, C.D., J.G. and G.M. provided important ideas and edited the manuscript, E.K. and K.M. designed experiments and wrote the manuscript.

Corresponding authors

Correspondence to Klaus Mohr or Evi Kostenis.

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Competing interests

The Epic biosensor was provided to E.K. by Corning, Inc.

Supplementary information

Supplementary Fig. 1

Solvent-induced DMR. Increasing concentrations of DMSO, which is frequently applied as solvent for ligands, were added to native HEK293 cells and the resulting alterations of cellular DMR are recorded. Note that DMSO concentrations greater than 0.1% trigger pronounced DMR alterations per se. Therefore compound solutions and DMR assay buffer should be carefully matched with respect to DMSO content to avoid spiky artificial solvent peaks. (EPS 95 kb)

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Schröder, R., Schmidt, J., Blättermann, S. et al. Applying label-free dynamic mass redistribution technology to frame signaling of G protein–coupled receptors noninvasively in living cells. Nat Protoc 6, 1748–1760 (2011). https://doi.org/10.1038/nprot.2011.386

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