Live Mitochondrial or Cytosolic Calcium Imaging Using Genetically-encoded Cameleon Indicator in Mammalian Cells

Elisa Greotti; Tullio Pozzan

doi:10.21769/BioProtoc.3504

Live Mitochondrial or Cytosolic Calcium Imaging Using Genetically-encoded Cameleon Indicator in Mammalian Cells

Bio Protoc. 2020 Feb 5;10(3):e3504. doi: 10.21769/BioProtoc.3504.

Authors

Elisa Greotti^{1

2}, Tullio Pozzan^{1

2

3}

Affiliations

¹ Neuroscience Institute, National Research Council (CNR), 35131 Padua, Italy.
² Department of Biomedical Sciences, University of Padua, 35131 Padua, Italy.
³ Venetian Institute of Molecular Medicine (VIMM), 35129 Padua, Italy.

Abstract

Calcium (Ca²⁺) imaging aims at investigating the dynamic changes in live cells of its concentration ([Ca²⁺]) in different pathophysiological conditions. Ca²⁺ is an ubiquitous and versatile intracellular signal that modulates a large variety of cellular functions thanks to a cell type-specific toolkit and a complex subcellular compartmentalization. Many Ca²⁺ sensors are presently available (chemical and genetically encoded) that can be specifically targeted to different cellular compartments. Using these probes, it is now possible to monitor Ca²⁺ dynamics of living cells not only in the cytosol but also within specific organelles. The choice of a specific sensor depends on the experimental design and the spatial and temporal resolution required. Here we describe the use of novel Förster resonance energy transfer (FRET)-based fluorescent Ca²⁺ probes to dynamically and quantitatively monitor the changes in cytosolic and mitochondrial [Ca²⁺] in a variety of cell types and experimental conditions. FRET-based sensors have the enormous advantage of being ratiometric, a feature that makes them particularly suitable for quantitative and in vivo applications.

Keywords: Cytosolic Ca2+ imaging; IP3 Receptor; mitochondrial Ca2+ imaging; Cameleon; Endoplasmic Reticulum; FRET; Genetically-Encoded Calcium Indicators; Mitochondria; Ryanodine Receptor.; SOCE.