High-throughput-compatible assays using a genetically-encoded calcium indicator

Nyantsz Wu,Michael P Maher,Walter K Nishioka,Noël C Derecki

doi:10.1038/s41598-019-49070-8

Nyantsz Wu, Michael P Maher + Show 2 more

Open Access

https://doi.org/10.1038/s41598-019-49070-8

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Journal: Scientific Reports	Publication Date: Sep 3, 2019
Citations: 26	License type: open-access

Affiliation: Janssen (United States)

Abstract

Measurement of intracellular calcium in live cells is a key component of a wide range of basic life science research, and crucial for many high-throughput assays used in modern drug discovery. Synthetic calcium indicators have become the industry standard, due their ease of use, high reliability, wide dynamic range, and availability of a large variety of spectral and chemical properties. Genetically-encoded calcium indicators (GECIs) have been optimized to the point where their performance rivals that of synthetic calcium indicators in many applications. Stable expression of a GECI has distinct advantages over synthetic calcium indicators in terms of reagent cost and simplification of the assay process. We generated a clonal cell line constitutively expressing GCaMP6s; high expression of the GECI was driven by coupling to a blasticidin resistance gene with a self-cleaving cis-acting hydrolase element (CHYSEL) 2A peptide. Here, we compared the performance of the GECI GCaMP6s to the synthetic calcium indicator fluo-4 in a variety of assay formats. We demonstrate that the pharmacology of ion channel and GPCR ligands as determined using the two indicators is highly similar, and that GCaMP6s is viable as a direct replacement for a synthetic calcium indicator.

Highlights

Calcium ions (Ca2+) serve as a ubiquitous second messenger within cells across all domains of life[1], modulating a wide array of cellular processes
For our initial characterization of GCaMP6s, we used flow cytometry to compare the fluorescence intensity of 293-F cells labelled with fluo-4 to cells 24 hours after transfecting with the pGP-CMV-GCaMP6s28 construct (Figure 1a–c)
Transient transfection of the genetically-encoded calcium indicator construct pGP-CMV-GCaMP6s into 293-F cells yielded a population of cells with a robust increase in fluorescence in the presence of ionomycin

Summary

Introduction

Calcium ions (Ca2+) serve as a ubiquitous second messenger within cells across all domains of life[1], modulating a wide array of cellular processes. Since the discovery of the first synthetic calcium indicators by Tsien[3], based upon the coupling of a fluorophore to a calcium chelator, multiple efforts to fine-tune affinity, selectivity, and optical properties have generated a large panel of probes (reviewed by Paredes et al.[4]). Their brightness, photostability, and large dynamic range have allowed synthetic calcium indicators to become the probes of choice for high-throughput calcium assays. Murayama et al used a GECI targeted to ER23 to screen for ryanodine receptor inhibitors[24]

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Conclusion