RESUMO
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.
