425 milliseconds of time interval, 371 milliseconds of time interval, 575 milliseconds of time interval, 100 milliseconds of time interval, 402 milliseconds of time interval
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Related paper(s)
Hiroko Bannai, Matsumi Hirose, Fumihiro Niwa, Katsuhiko Mikoshiba (2019) Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators., Journal of visualized experiments : JoVE, Number 145
Published in 2019 Mar 22
(Electronic publication in March 22, 2019, midnight )
(Abstract) Calcium ion (Ca(2+)) is a universal intracellular messenger molecule that drives multiple signaling pathways, leading to diverse biological outputs. The coordination of two Ca(2+) signal sources-"Ca(2+) influx" from outside the cell and "Ca(2+) release" from the intracellular Ca2+ store endoplasmic reticulum (ER)-is considered to underlie the diverse spatio-temporal patterns of Ca(2+) signals that cause multiple biological functions in cells. The purpose of this protocol is to describe a new Ca(2+) imaging method that enables monitoring of the very moment of "Ca(2+) influx" and "Ca(2+) release". OER-GCaMP6f is a genetically encoded Ca(2+) indicator (GECI) comprising GCaMP6f, which is targeted to the ER outer membrane. OER-GCaMP6f can monitor Ca(2+) release at a higher temporal resolution than conventional GCaMP6f. Combined with plasma membrane-targeted GECIs, the spatio-temporal Ca(2+) signal pattern can be described at a subcellular resolution. The subcellular-targeted Ca(2+) 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 Ca(2+) 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.