PMID- 31481721
OWN - NLM
STAT- MEDLINE
DCOM- 20201020
LR  - 20210110
IS  - 2045-2322 (Electronic)
IS  - 2045-2322 (Linking)
VI  - 9
IP  - 1
DP  - 2019 Sep 3
TI  - High-throughput-compatible assays using a genetically-encoded calcium indicator.
PG  - 12692
LID - 10.1038/s41598-019-49070-8 [doi]
LID - 12692
AB  - 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.
FAU - Wu, Nyantsz
AU  - Wu N
AD  - Janssen Research & Development, LLC, San Diego, CA, 92121, USA.
FAU - Nishioka, Walter K
AU  - Nishioka WK
AD  - Janssen Research & Development, LLC, San Diego, CA, 92121, USA.
FAU - Derecki, Noel C
AU  - Derecki NC
AD  - Janssen Research & Development, LLC, San Diego, CA, 92121, USA.
FAU - Maher, Michael P
AU  - Maher MP
AUID- ORCID: 0000-0001-9138-0448
AD  - Janssen Research & Development, LLC, San Diego, CA, 92121, USA. 
      mmaher1@its.jnj.com.
LA  - eng
PT  - Comparative Study
PT  - Journal Article
DEP - 20190903
PL  - England
TA  - Sci Rep
JT  - Scientific reports
JID - 101563288
RN  - 0 (Aniline Compounds)
RN  - 0 (Calmodulin)
RN  - 0 (Fluo 4)
RN  - 0 (Receptors, G-Protein-Coupled)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (Xanthenes)
RN  - SY7Q814VUP (Calcium)
SB  - IM
MH  - Aniline Compounds/chemistry
MH  - *Biological Assay
MH  - Calcium/*analysis/metabolism
MH  - *Calcium Signaling
MH  - Calmodulin/genetics/metabolism
MH  - HEK293 Cells
MH  - Humans
MH  - Receptors, G-Protein-Coupled/genetics/metabolism
MH  - Recombinant Fusion Proteins/genetics/metabolism
MH  - Xanthenes/chemistry
PMC - PMC6722131
COIS- At the time of the work described in this manuscript, all authors were employees 
      of Janssen Research & Development, LLC, a division of Johnson & Johnson. All 
      authors own stock in Johnson & Johnson.
EDAT- 2019/09/05 06:00
MHDA- 2020/10/21 06:00
PMCR- 2019/09/03
CRDT- 2019/09/05 06:00
PHST- 2019/04/05 00:00 [received]
PHST- 2019/08/19 00:00 [accepted]
PHST- 2019/09/05 06:00 [entrez]
PHST- 2019/09/05 06:00 [pubmed]
PHST- 2020/10/21 06:00 [medline]
PHST- 2019/09/03 00:00 [pmc-release]
AID - 10.1038/s41598-019-49070-8 [pii]
AID - 49070 [pii]
AID - 10.1038/s41598-019-49070-8 [doi]
PST - epublish
SO  - Sci Rep. 2019 Sep 3;9(1):12692. doi: 10.1038/s41598-019-49070-8.