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

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.