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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.

Dissection of Local Ca<sup>2+</sup> Signals in Cultured Cells by Membrane-targeted Ca<sup>2+</sup> Indicators