The endoplasmic reticulum (ER), the major organelle for the storage of Ca2+, maintains a concentration of Ca2+ much higher than in the cytosol or other subcellular organelles, such as the mitochondria. A variety of tools have been developed for measuring Ca2+ activity in neuronal and glial cells, but most of these sensors target either the plasma membrane (PM) or the cytosol. Though these sensors are important for measuring Ca2+ transients, they lack the capability to measure activity in the periphery of the ER or to measure low-amplitude events resulting from Ca2+ exchange between the ER and other organelles, such as the mitochondria. We recently developed an ER-targeted GCaMP6f anchored to the cytosolic side of the ER that can measure minute calcium exchange occurring in this region. In this article, we discuss detailed methods to characterize the ER-GCaMP6f sensor, utilize it for calcium imaging in cultured astrocytes, and assess its expression and calcium imaging in astrocytes in rodent brains.
Bibliographical noteFunding Information:
The authors acknowledge the Genetic Technologies Core at the Center of Molecular Medicine and Light Microscopy Core, University of Kentucky, for use of their facilities. CIR acknowledges support from the National Institutes of Health (DA038817). Cartoons were constructed with BioRender (biorender.com). Chemical structures were drawn with the help of ChemSketch.
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ASJC Scopus subject areas
- Neuroscience (all)
- Biochemistry, Genetics and Molecular Biology (all)
- Immunology and Microbiology (all)
- Pharmacology, Toxicology and Pharmaceutics (all)
- Health Informatics
- Medical Laboratory Technology