Staff Publications

Staff Publications

  • external user (warningwarning)
  • Log in as
  • language uk
  • About

    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

    We have a manual that explains all the features 

Record number 534476
Title Calcium imaging of GPCR activation using arrays of reverse transfected HEK293 cells in a microfluidic system
Author(s) Roelse, Margriet; Henquet, Maurice G.L.; Verhoeven, Harrie A.; Ruijter, Norbert C.A. De; Wehrens, Ron; Lenthe, Marco S. Van; Witkamp, Renger F.; Hall, Robert D.; Jongsma, Maarten A.
Source Sensors 18 (2018)2. - ISSN 1424-8220
DOI https://doi.org/10.3390/s18020602
Department(s) PRI BIOS Applied Metabolic Systems
Laboratory of Cell Biology
Biometris (WU MAT)
Biometris (PPO/PRI)
Chair Nutrition and Pharmacology (HNE)
VLAG
Publication type Refereed Article in a scientific journal
Publication year 2018
Keyword(s) Cameleon YC3.6 - Cell array - GPCR - Microfluidics - NK1 receptor - Reverse transfection
Abstract Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries.
Comments
There are no comments yet. You can post the first one!
Post a comment
 
Please log in to use this service. Login as Wageningen University & Research user or guest user in upper right hand corner of this page.