Cellular Test System with Genetically Encoded Indicators of Cytosolic and Reticular Са<sup>2+</sup>
Calcium ions are a universal second messenger that regulates numerous intracellular processes. Many agonists and first messengers involved in cell-to-cell communications and autocrine/paracrine regulation act through surface receptors coupled to the mobilization of intracellular Са<sup>2+</sup>. The current concept of intracellular Са<sup>2+</sup> signaling is mainly based on monitoring of Са<sup>2+</sup> in the cytosol. However, the Са<sup>2+</sup> exchange between the cytosol and a variety of intracellular organelles plays an important role in cell and tissue physiology. Therefore, monitoring of Са<sup>2+</sup> signals in cell compartments is obligatory for uncovering key aspects of intracellular Са<sup>2+</sup> signaling. Until recently, intracellular Са<sup>2+</sup> signaling was studied using synthetic Са<sup>2+</sup> probes, which cannot be selectively loaded into cell compartments involved in Са<sup>2+</sup> homeostasis, including the cytosol, endoplasmic reticulum, mitochondria, and lysosomes. Therefore, one tried to identify Са<sup>2+</sup> signals specific for compartments by using Са<sup>2+</sup> probes with different affinities in combination with the permeabilization of the plasma membrane. In the last decade, an array of Са<sup>2+</sup> sensitive fluorescent proteins have been engineered and used as genetically encoded Са<sup>2+</sup> indicators. One of the main advantages of these indicators is that they can be selectively expressed and retained in certain organelles. In this article we describe the elaboration of monoclonal cell lines, which functionally express both fluorescent sensors of cytosol Са<sup>2+</sup> GEM-GECO1 and reticular Са<sup>2+</sup> R-CEPIA1er. The obtained cell clones can be used as a test system for the analysis of the coupling of surface receptors to the intracellular Са<sup>2+</sup> signaling and for studying mechanisms controlling Са<sup>2+</sup> release.