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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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).
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.
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Agonist-induced activation of human FFA1 receptor signals to extracellular signal-regulated kinase 1 and 2 through Gq- and Gi-coupled signaling cascades
Cellular & Molecular Biology Letters (2017)