Practical aspects of measuring intracellular calcium signals with fluorescent indicators.

The use of fluorescent indicators for monitoring calcium (Ca(2+)) signals and for measuring Ca(2+) concentration ([Ca(2+)]) in living cells is described. The following topics are covered in detail: (1) ratiometric and nonratiometric fluorescent indicators and the principles underlying their use, (2) techniques for loading Ca(2+) indicators and Ca(2+) buffers into living cells, (3) calibration of indicator fluorescence intensity measurements to yield values of intracellular [Ca(2+)], (4) analysis of nonratiometric fluorescence intensity data and caveats relating to their interpretation, (5) techniques for manipulating intracellular and extracellular [Ca(2+)], and (6) the use of fluorescent indicators to monitor Ca(2+) signals in mitochondria. The chapter aims to present these fundamental topics in a manner that is practically useful and intuitively accessible. The origins of key mathematical equations used in the article are outlined in two appendices.

[1]  W. Webb,et al.  A confocal laser scanning microscope designed for indicators with ultraviolet excitation wavelengths. , 1994, The American journal of physiology.

[2]  B. Hille,et al.  Mitochondrial Participation in the Intracellular Ca2+ Network , 1997, The Journal of cell biology.

[3]  P. Mcneil.  Direct Introduction of Molecules into Cells , 2003, Current protocols in cell biology.

[4]  Arthur E. Martell,et al.  Critical Stability Constants , 2011 .

[5]  A Kusumi,et al.  Comparison of two‐photon excitation laser scanning microscopy with UV‐confocal laser scanning microscopy in three‐dimensional calcium imaging using the fluorescence indicator Indo‐1 , 1997, Journal of microscopy.

[6]  Brian J. Bacskai,et al.  Video-Rate Confocal Microscopy , 1995 .

[7]  R. Tsien,et al.  Cytosolic Ca2+ homeostasis in Ehrlich and Yoshida carcinomas. A new, membrane-permeant chelator of heavy metals reveals that these ascites tumor cell lines have normal cytosolic free Ca2+. , 1985, The Journal of biological chemistry.

[8]  R Y Tsien,et al.  Photochemically generated cytosolic calcium pulses and their detection by fluo-3. , 1989, The Journal of biological chemistry.

[9]  R Y Tsien,et al.  Calcium homeostasis in intact lymphocytes: cytoplasmic free calcium monitored with a new, intracellularly trapped fluorescent indicator , 1982, The Journal of cell biology.

[10]  R. Tsien,et al.  Active involvement of Ca2+ in mitotic progression of Swiss 3T3 fibroblasts , 1990, The Journal of cell biology.

[11]  R Y Tsien,et al.  New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis, and properties of prototype structures. , 1980, Biochemistry.

[12]  C Liu,et al.  Characterization of ionomycin as a calcium ionophore. , 1978, The Journal of biological chemistry.

[13]  F. Di Virgilio,et al.  Inhibition of Fura-2 sequestration and secretion with organic anion transport blockers. , 1990, Cell calcium.

[14]  P. Mcneil,et al.  Incorporation of macromolecules into living cells. , 1989, Methods in cell biology.

[15]  R. Tsien,et al.  Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. , 1989, The Journal of biological chemistry.

[16]  Richard P. Haugland,et al.  Handbook of fluorescent probes and research chemicals , 1996 .

[17]  G. Anderegg,et al.  Pyridinderivate als Komplexbildner. XI†. Die Thermodynamik der Metallkomplexbildung mit Bis-, Tris- und Tetrakis[(2-pyridyl)methyl]-aminen , 1977 .

[18]  R. Tsien,et al.  [14] Measurement of cytosolic free Ca2+ with quin2 , 1989 .

[19]  J. Meldolesi,et al.  Fura-2 measurement of cytosolic free Ca2+ in monolayers and suspensions of various types of animal cells , 1987, The Journal of cell biology.

[20]  R. Tsien,et al.  A new generation of Ca2+ indicators with greatly improved fluorescence properties. , 1985, The Journal of biological chemistry.

[21]  R. Hotchkiss,et al.  Effect of Ca2+ agonists in the perfused liver: determination via laser scanning confocal microscopy. , 1999, The American journal of physiology.

[22]  F. Di Virgilio,et al.  Fura-2 secretion and sequestration in macrophages. A blocker of organic anion transport reveals that these processes occur via a membrane transport system for organic anions. , 1988, Journal of immunology.

[23]  J. Pawley,et al.  Handbook of Biological Confocal Microscopy , 1990, Springer US.

[24]  G. Anderegg,et al.  Pyridinderivate als Komplexbildner VIII Die Herstellung je eines neuen vier‐ und sechszähnigen Liganden , 1967 .

[25]  D. Callaham,et al.  Free calcium increases during anaphase in stamen hair cells of Tradescantia , 1987, The Journal of cell biology.