Alba Delrio-lorenzo , Jonathan Rojo-ruiz, Patricia Torres-vidal, Maria Teresa Alonso, Javier García-sancho
{"title":"In vitro and in vivo calibration of low affinity genetic Ca2+ indicators","authors":"Alba Delrio-lorenzo , Jonathan Rojo-ruiz, Patricia Torres-vidal, Maria Teresa Alonso, Javier García-sancho","doi":"10.1016/j.ceca.2023.102819","DOIUrl":null,"url":null,"abstract":"<div><p>Calcium is a universal intracellular messenger and proper Ca<sup>2+</sup>concentrations ([C<em>a</em><sup>2+</sup>]) both in the cytosol and in the lumen of cytoplasmic organelles are essential for cell functions. Ca<sup>2+</sup> homeostasis is achieved by a delicate pump/leak balance both at the plasma membrane and at the endomembranes, and improper Ca<sup>2+</sup> levels result in malfunction and disease. Selective intraorganellar Ca<sup>2+</sup>measurements are best achieved by using targeted genetically encoded Ca<sup>2+</sup> indicators (GECIs) but to calibrate the luminal fluorescent signals into accurate [Ca<sup>2+</sup>] is challenging, especially <em>in vivo</em>, due to the difficulty to normalize and calibrate the fluorescent signal in various tissues or conditions. We report here a procedure to calibrate the ratiometric signal of GAP (GFP-Aequorin Protein) targeted to the endo-sarcoplasmic reticulum (ER/SR) into [Ca<sup>2+</sup>]<sub>ER/SR</sub> based on imaging of fluorescence after heating the tissue at 50–52 °C, since this value coincides with that obtained in the absence of Ca<sup>2+</sup> (R<sub>min</sub>). Knowledge of the dynamic range (R<sub>max</sub>/R<sub>min</sub>) and the Ca<sup>2+</sup>-affinity (K<sub>D</sub>) of the indicator permits calculation of [Ca<sup>2+</sup>] by applying a simple algorithm. We have validated this procedure <em>in vitro</em> using several cell types (HeLa, HEK 293T and mouse astrocytes), as well as <em>in vivo</em> in <em>Drosophila</em>. Moreover, this methodology is applicable to other low Ca<sup>2+</sup> affinity green and red GECIs.</p></div>","PeriodicalId":9678,"journal":{"name":"Cell calcium","volume":null,"pages":null},"PeriodicalIF":4.3000,"publicationDate":"2023-11-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Cell calcium","FirstCategoryId":"99","ListUrlMain":"https://www.sciencedirect.com/science/article/pii/S0143416023001306","RegionNum":2,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CELL BIOLOGY","Score":null,"Total":0}
引用次数: 0
Abstract
Calcium is a universal intracellular messenger and proper Ca2+concentrations ([Ca2+]) both in the cytosol and in the lumen of cytoplasmic organelles are essential for cell functions. Ca2+ homeostasis is achieved by a delicate pump/leak balance both at the plasma membrane and at the endomembranes, and improper Ca2+ levels result in malfunction and disease. Selective intraorganellar Ca2+measurements are best achieved by using targeted genetically encoded Ca2+ indicators (GECIs) but to calibrate the luminal fluorescent signals into accurate [Ca2+] is challenging, especially in vivo, due to the difficulty to normalize and calibrate the fluorescent signal in various tissues or conditions. We report here a procedure to calibrate the ratiometric signal of GAP (GFP-Aequorin Protein) targeted to the endo-sarcoplasmic reticulum (ER/SR) into [Ca2+]ER/SR based on imaging of fluorescence after heating the tissue at 50–52 °C, since this value coincides with that obtained in the absence of Ca2+ (Rmin). Knowledge of the dynamic range (Rmax/Rmin) and the Ca2+-affinity (KD) of the indicator permits calculation of [Ca2+] by applying a simple algorithm. We have validated this procedure in vitro using several cell types (HeLa, HEK 293T and mouse astrocytes), as well as in vivo in Drosophila. Moreover, this methodology is applicable to other low Ca2+ affinity green and red GECIs.
期刊介绍:
Cell Calcium covers the field of calcium metabolism and signalling in living systems, from aspects including inorganic chemistry, physiology, molecular biology and pathology. Topic themes include:
Roles of calcium in regulating cellular events such as apoptosis, necrosis and organelle remodelling
Influence of calcium regulation in affecting health and disease outcomes