[Methods in Cell Biology] Calcium in Living Cells Volume 99 || Practical Aspects of Measuring Intracellular Calcium Signals with Fluorescent Indicators

✍ Scribed by Kao, Joseph P.Y.

• Book ID: 120984889
• Publisher: Elsevier
• Year: 2010
• Tongue: English
• Weight: 968 KB
• Edition: 2
• Category: Article
• ISBN: 0123748410
• ISSN: 0091-679X
• DOI: 10.1016/B978-0-12-374841-6.00005-0

✦ Synopsis

I. Introduction II. Fluorescent Ca 2þ Indicators III. Loading Indicators into Cells A. Limited Aqueous Solubility of AM Esters B. Dye Compartmentalization: Loading of Indicator into Subcellular Compartments Other than the Cytosol C. Dye Leakage or Extrusion from Cells D. Procedure for Loading IV. Manipulation of [Ca 2þ ] A. Using EGTA and BAPTA as Extracellular Ca 2þ BuVers B. Lowering Extracellular [Ca 2þ ] C. Divalent Cation Ionophores D. BuVering Changes in Intracellular [Ca 2þ ] V. Conversion of Indicator Fluorescence Signal into Values of [Ca 2þ ] A. Calibrating a Nonratiometric Fluorescent Indicator B. Calibrating a Ratiometric Fluorescent Indicator VI. Reporting Indicator Fluorescence Intensity Changes without Calibration A. Reporting Relative Changes in Fluorescence: F/F 0 and DF/F 0 B. Caveat in Interpreting Relative Fluorescence Changes: Indicator Fluorescence is Not a Linear Function of [Ca 2þ ] VII. Measuring [Ca 2þ ] in Mitochondria A. Estimating the Fraction of Intracellular Rhod-2 Indicator that Resides in Mitochondria B. Minimizing Rhod-2 Loading in the Cytosol C. Monitoring Cytosolic and Mitochondrial [Ca 2þ ] Simultaneously