Dissection of Local Ca2+ Signals in Cultured Cells by Membrane-targeted Ca2+ Indicators
| Autor: | Matsumi Hirose, Hiroko Bannai, Katsuhiko Mikoshiba, Fumihiro Niwa |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Cell type 030102 biochemistry & molecular biology General Immunology and Microbiology Chemistry Endoplasmic reticulum General Chemical Engineering General Neuroscience Cell General Biochemistry Genetics and Molecular Biology Cell biology 03 medical and health sciences 030104 developmental biology medicine.anatomical_structure medicine Neuron Signal transduction Intracellular Astrocyte Calcium signaling |
| Zdroj: | Journal of Visualized Experiments. |
| ISSN: | 1940-087X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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