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

Margriet Roelse ,Robert D Hall ,Renger F Witkamp ,Maarten A Jongsma ,Harrie A Verhoeven ,Marco S Van Lenthe ,Ron Wehrens ,Maurice G.l Henquet ,Norbert C.a De Ruijter

doi:10.3390/s18020602

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.

Highlights

Reverse transfection is a technique to create arrays of transiently-transfected cells on a solid surface
We focused on the parameters of the reverse transfection procedure to optimize G protein-coupled receptor (GPCR) response in our microfluidic system and on the appropriate data analysis
This paper has studied the main parameters for optimization of reverse transfection cell arrays for the detection of GPCR signaling in a microfluidic system

Summary

Introduction

Reverse transfection is a technique to create arrays of transiently-transfected cells on a solid surface. Steps (i) and (ii) are sometimes combined [4,8,9], but typically, these three basic steps can transform a printed DNA microarray into a transfected cell array via efficient contact-mediated uptake of plasmid DNA by the adhering cells. This is visualized, Steps 1 and 2, Video S1 and Figure S1. To a great extent, on correct formation and delivery of DNA transfection complexes These aspects have been studied for both surface-immobilized and solution-mediated transfection systems [10,11,12]. Cohen et al [15] have shown that with increasing DNA levels, the maximum gene expression depends on cell type, the vector used and the method of transfection, so that no single protocol can be applied in all situations

Results

Discussion

Conclusion