Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System

Sensors
Margriet RoelseMaarten A Jongsma

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 1...Continue Reading

References

Nov 15, 1990·Analytical Biochemistry·M G KauffmanA D Donnenberg
Nov 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·P L FelgnerM Danielsen
Sep 1, 1995·General Pharmacology·C A Maggi
Aug 11, 1995·The Journal of Biological Chemistry·J ZabnerM J Welsh
Jan 1, 1995·Methods in Enzymology·W Krek, J A DeCaprio
May 3, 2001·Nature·J Ziauddin, D M Sabatini
Jun 22, 2001·BMC Chemical Biology·Vicki J Bennett, Mark A Simmons
May 14, 2004·Journal of Biomolecular Screening·Yuji M MishinaCraig M Thompson
Jul 13, 2004·Proceedings of the National Academy of Sciences of the United States of America·Takeharu NagaiAtsushi Miyawaki
Nov 10, 2004·Proceedings of the National Academy of Sciences of the United States of America·Steve N BaileyBrent R Stockwell
Dec 20, 2005·Journal of Controlled Release : Official Journal of the Controlled Release Society·Rumiko MoriguchiHideyoshi Harashima
Jan 25, 2006·Nature Methods·Steve N BaileyDavid M Sabatini
Oct 20, 2006·Journal of Nanoscience and Nanotechnology·Carsten KneuerUdo Bakowsky
Sep 27, 2008·Lab on a Chip·Reiko Onuki-NagasakiJun Miyake
Jan 17, 2009·Biotechnology and Bioengineering·Zain BengaliLonnie D Shea
Feb 13, 2009·Journal of Controlled Release : Official Journal of the Controlled Release Society·Richard N CohenFrancis C Szoka
Apr 22, 2009·Molecular BioSystems·Masato MiyakeJun Miyake
Dec 22, 2009·Chemical Senses·Wolfgang MeyerhofMaik Behrens
Jun 4, 2010·Lab on a Chip·Jun TianStelios T Andreadis
Jan 22, 2011·Trends in Biotechnology·Amy E PalmerJanet E McCombs
Jan 31, 2013·Lab on a Chip·Benjamin R SchudelAnup K Singh
Sep 3, 2013·Journal of Pharmaceutical Sciences·Jonathan De La VegaDuarte Miguel F Prazeres
Oct 10, 2013·Biosensors & Bioelectronics·Eun Hae OhTai Hyun Park
Nov 1, 2012·Wiley Interdisciplinary Reviews. Membrane Transport and Signaling·Laurie Erb, Gary A Weisman
Aug 8, 2015·PeerJ·Seyed Mohammad Ali HaghparastJun Miyake
Apr 1, 2016·Scientific Reports·Kristina Woodruff, Sebastian J Maerkl
Jul 12, 2016·Metabolomics : Official Journal of the Metabolomic Society·M G L HenquetM A Jongsma

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Methods Mentioned

BETA
transfection
FRET
PCR
Chip
Profiler
DNA array
Fluorescence

Software Mentioned

CellProfiler
house
Cell Profiler
MetaMorph Imaging Series
LAS AF Lite
Leica
in

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