Abstract Quantitative assessment of cell migration in vitro is often required in fundamental and applied research from different biomedical areas including wound repair, tumor metastasis or developmental biology. A collection of assays has been established throughout the years like the most widely used scratch assay or the so-called barrier assay . It is the principle of these assays to introduce a lesion into an otherwise confluent monolayer in order to study the migration of cells from the periphery into this artificial wound and determine the migration rate from the time necessary for wound closure. A novel assay makes use of photosensitizers doped into a polystyrene matrix. A thin layer of this composite material is coated on the bottom of regular cell culture ware showing perfect biocompatibility. When adherent cells are grown on this coating, resonant excitation of the photosensitizer induces a very local generation of 1 O 2 , which kills the cells residing at the site of illumination. Cells outside the site of illumination are not harmed. When excitation of the photosensitizer is conducted by microscopic illumination, high-precision wounding in any size and geometry is available even in microfluidic channels. Besides proof-of-concept experiments, this study gives further insight into the mechanism of photosensitizer-mediated cell wounding.
Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample's impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).
G protein-coupled cell surface receptors (GPCR) trigger complex intracellular signaling cascades upon agonist binding. Classic pharmacological assays provide information about binding affinities, activation or blockade at different stages of the signaling cascade, but real time dynamics and reversibility of these processes remain often disguised. We show that combining photochromic NPY receptor ligands, which can be toggled in their receptor activation ability by irradiation with light of different wavelengths, with whole cell label-free impedance assays allows observing the cell response to receptor activation and its reversibility over time. The concept demonstrated on NPY receptors may be well applicable to many other GPCRs providing a deeper insight into the time course of intracellular signaling processes.
Abstract G protein‐coupled cell surface receptors (GPCR) trigger complex intracellular signaling cascades upon agonist binding. Classic pharmacological assays provide information about binding affinities, activation or blockade at different stages of the signaling cascade, but real time dynamics and reversibility of these processes remain often disguised. We show that combining photochromic NPY receptor ligands, which can be toggled in their receptor activation ability by irradiation with light of different wavelengths, with whole cell label‐free impedance assays allows observing the cell response to receptor activation and its reversibility over time. The concept demonstrated on NPY receptors may be well applicable to many other GPCRs providing a deeper insight into the time course of intracellular signaling processes.
Abstract Sämtliche Polizeigesetze enthalten sog. Privatrechtsklauseln, die das durch den Gewaltenteilungsgrundsatz determinierte Verhältnis der Polizei zu den ordentlichen Gerichten ausformen. Viele der sich in diesem Kontext stellenden Rechtsfragen sind bislang nur ansatzweise in das Bewusstsein von Forschung und Rechtsanwendung getreten, weshalb die Untersuchung in weiten Teilen gleichsam dogmatisches Neuland betritt. So wird im Rahmen einer eingehenden Analyse die Unvereinbarkeit der herrschenden Auslegung der Vorschriften mit deren ratio legis aufgezeigt und dargelegt, dass aus teleologischen Gründen unter den dort genannten privaten Rechten ›sämtliche subjektive Rechte‹, nicht aber ›subjektive Rechtsgüter‹ zu verstehen sind. Zudem erfahren die in den Privatrechtsklauseln normierten – aufgrund ihrer zivilrechtlichen Implikationen teilweise hochkomplexen – Voraussetzungen für ein polizeiliches Eingreifen sowie die sich aus den Vorschriften ergebenden Rechtsfolgen eine detaillierte Betrachtung.»The Authority of the Police to Protect Private Rights. An Analysis of the So-Called Private Rights Clauses in Federal and State Police Laws«: All police laws contain so-called private right clauses that shape the relationship of the police to the civil courts. Many of the legal questions that arise in this context have so far only rudimentarily entered the consciousness of researchers and practitioners, which is in large part why the study breaks new ground in the field. In particular, an in-depth analysis reveals the incompatibility of the prevailing interpretation of the norms with their ratio legis.
Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample's impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).
Journal Article Increasing the throughput of label-free cell assays to study the activation of G-protein-coupled receptors by using a serial agonist exposure protocol Get access J A Stolwijk, J A Stolwijk Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, Germany Search for other works by this author on: Oxford Academic Google Scholar M Skiba, M Skiba Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, Germany Search for other works by this author on: Oxford Academic Google Scholar C Kade, C Kade Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, Germany Search for other works by this author on: Oxford Academic Google Scholar G Bernhardt, G Bernhardt Institute of Pharmacy, University of Regensburg, Regensburg, Germany Search for other works by this author on: Oxford Academic Google Scholar A Buschauer, A Buschauer Institute of Pharmacy, University of Regensburg, Regensburg, Germany Search for other works by this author on: Oxford Academic Google Scholar H Hübner, H Hübner Department of Chemistry and Pharmacy, Friedrich-Alexander University Erlangen-Nürnberg FAU, Erlangen, Germany Search for other works by this author on: Oxford Academic Google Scholar P Gmeiner, P Gmeiner Department of Chemistry and Pharmacy, Friedrich-Alexander University Erlangen-Nürnberg FAU, Erlangen, Germany Search for other works by this author on: Oxford Academic Google Scholar J Wegener J Wegener Institute of Analytical Chemistry, Chemo- and Biosensors, University of Regensburg, Regensburg, GermanyFraunhofer Research Institution for Microsystems and Solid State Technologies EMFT, Munich, Germany Corresponding author. E-mail: joachim.wegener@ur.de Search for other works by this author on: Oxford Academic Google Scholar Integrative Biology, Volume 11, Issue 3, March 2019, Pages 99–108, https://doi.org/10.1093/intbio/zyz010 Published: 13 May 2019 Article history Received: 23 January 2019 Revision received: 12 March 2019 Accepted: 03 May 2019 Published: 13 May 2019
