Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators

Hiroko Bannai*, Matsumi Hirose, Fumihiro Niwa, Katsuhiko Mikoshiba

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

4 Citations (Scopus)


Calcium ion (Ca2+) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca2+ signal sources—“Ca2+ influx” from outside the cell and “Ca2+ release” from the intracellular Ca2+ store endoplasmic reticulum (ER)—is considered to underlie the diverse spatio-temporal patterns of Ca2+ signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca2+ imaging method that enables monitoring of the very moment of “Ca2+ influx” and “Ca2+ release”. OER-GCaMP6f is a genetically encoded Ca2+ indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca2+ release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca2+ signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca2+ indicators described here are, in principle, available for all cell types, even for the in vivo imaging of Caenorhabditis elegans neurons. In this protocol, we introduce Ca2+ imaging in cells from cell lines, neurons, and glial cells in dissociated primary cultures, and describe the preparation of frozen stock of rat cortical neurons.

Original languageEnglish
Article numbere59246
JournalJournal of Visualized Experiments
Issue number145
Publication statusPublished - 2019 Mar
Externally publishedYes


  • Biological Science Disciplines
  • Biology
  • Ca imaging
  • Ca influx
  • Ca release
  • Cell Biology
  • Disciplines and Occupations
  • GCaMP6f
  • Issue 145
  • Local Ca
  • Natural Science Disciplines
  • Neurobiology
  • Neuroscience
  • Neurosciences
  • RCaMP2
  • astrocyte
  • cell line
  • dissociated culture
  • endoplasmic reticulum
  • neuron
  • plasma membrane

ASJC Scopus subject areas

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)


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