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

• Published in: Scientific reports Vol. 9; no. 1; pp. 12692 - 17
• Main Authors: Wu, Nyantsz, Nishioka, Walter K, Derecki, Noël C, Maher, Michael P
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 03.09.2019; Nature Publishing Group UK
• Subjects: Aniline Compounds - chemistry; Biological Assay; Calcium (intracellular); Calcium - analysis; Calcium - metabolism; Calcium Signaling; Calmodulin - genetics; Calmodulin - metabolism; Cytotoxicity; G protein-coupled receptors; HEK293 Cells; Humans; Hydrolase; Molecular biology; Optimization; Peptides; Proteins; R&D; Receptors, G-Protein-Coupled - genetics; Receptors, G-Protein-Coupled - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Research & development; Xanthenes - chemistry
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-019-49070-8

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.