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. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Expression and characterization of ER-GCaMP6f Support Protocol 1: ER-GCaMP6f-expressing stable cell line generation Basic Protocol 2: In vitro calcium imaging with ER-GCaMP6f Support Protocol 2: Imaging of drug-induced calcium activity Alternate Protocol 1: Transduction of astrocytes with ER-GCaMP6f AAV Alternate Protocol 2: Calcium imaging of astrocytes with Fluo-4 AM Basic Protocol 3: In vivo ER-GCaMP6f expression and slice calcium imaging Support Protocol 3: Pharmacological studies with 2-APB in brain slices.
Keywords: AAV; ER-GCaMP6f; astrocyte; calcium; fluorescence.
© 2022 Wiley Periodicals LLC.