Department of Chemistry, University of Kentucky, Lexington, Kentucky.
Department of Neuroscience, University of Kentucky, Lexington, Kentucky.
Curr Protoc. 2022 Aug;2(8):e491. doi: 10.1002/cpz1.491.
The endoplasmic reticulum (ER), the major organelle for the storage of Ca , maintains a concentration of Ca much higher than in the cytosol or other subcellular organelles, such as the mitochondria. A variety of tools have been developed for measuring Ca 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 Ca transients, they lack the capability to measure activity in the periphery of the ER or to measure low-amplitude events resulting from Ca 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.
内质网（ER）是储存 Ca 的主要细胞器，其 Ca 浓度远高于细胞质或其他亚细胞器，如线粒体。已经开发出多种工具来测量神经元和神经胶质细胞中的 Ca 活性，但这些传感器中的大多数都针对质膜（PM）或细胞质。虽然这些传感器对于测量 Ca 瞬变很重要，但它们缺乏测量 ER 外围活性的能力，也无法测量 ER 与其他细胞器（如线粒体）之间 Ca 交换导致的低幅度事件。我们最近开发了一种 ER 靶向的 GCaMP6f，它锚定在 ER 的细胞质侧，可以测量该区域发生的微小钙交换。在本文中，我们讨论了表征 ER-GCaMP6f 传感器的详细方法，将其用于培养的星形胶质细胞中的钙成像，并评估其在啮齿动物大脑中星形胶质细胞中的表达和钙成像。© 2022 威立出版社。基础方案 1：ER-GCaMP6f 的表达和表征支持方案 1：ER-GCaMP6f 表达稳定细胞系的生成基础方案 2：使用 ER-GCaMP6f 进行体外钙成像支持方案 2：药物诱导钙活性的成像备选方案 1：用 ER-GCaMP6f AAV 转导星形胶质细胞备选方案 2：使用 Fluo-4 AM 进行星形胶质细胞钙成像基础方案 3：体内 ER-GCaMP6f 表达和切片钙成像支持方案 3：在脑片中使用 2-APB 的药理学研究。
