Calcium imaging of GPCR activation using arrays of reverse transfected HEK293 cells in a microfluidic system

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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.
Original languageEnglish
Article number602
JournalSensors (Switzerland)
Volume18
Issue number2
DOIs
Publication statusPublished - 16 Feb 2018

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Microfluidics
HEK293 Cells
G-Protein-Coupled Receptors
calcium
Calcium
DNA
Chemical activation
activation
proteins
Proteins
Imaging techniques
deoxyribonucleic acid
cells
Neurokinin-1 Receptors
Gene expression
Transfection
Screening
Plasmids
plasmids
gene expression

Keywords

  • Cameleon YC3.6
  • Cell array
  • GPCR
  • Microfluidics
  • NK1 receptor
  • Reverse transfection

Cite this

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title = "Calcium imaging of GPCR activation using arrays of reverse transfected HEK293 cells in a microfluidic system",
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.",
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author = "Margriet Roelse and Henquet, {Maurice G.L.} and Verhoeven, {Harrie A.} and {De Ruijter}, {Norbert C.A.} and Ron Wehrens and {Van Lenthe}, {Marco S.} and Witkamp, {Renger F.} and Hall, {Robert D.} and Jongsma, {Maarten A.}",
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AU - Wehrens, Ron

AU - Van Lenthe, Marco S.

AU - Witkamp, Renger F.

AU - Hall, Robert D.

AU - Jongsma, Maarten A.

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