Simultaneous analysis of intracellular pH and Ca<sup>2+</sup> from cell populations

Raul Martinez-Zaguilan, Linda S. Tompkins, Robert J. Gillies, Ron Lynch

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

Abstract

Although changes in both pH<inf>in</inf> and [Ca<sup>2+</sup>]<inf>i</inf> have been observed in response to a variety of agonists, it is not clear whether these ionic events work independently or are coordinated to lead to a specific physiological response. One of the fundamental problems in studying these ionic events is that changes in pH<inf>in</inf> modify Ca<sup>2+</sup> regulatory mechanisms and changes in Ca<sup>2+</sup> may modify pH regulation. It is desirable to use a technique that allows concomitant monitoring of these two ions in cell populations with high time resolution. Furthermore, like many Ca<sup>2+</sup> binding proteins, all Ca<sup>2+</sup> -sensitive fluoroprobes are inherently sensitive to pH owing to competition of H<sup>+</sup> for the Ca<sup>2+</sup> -binding sites. This chapter describes experimental paradigms that provide optimum conditions for simultaneous measurement of pH from the fluorescence emission of snarf-1, and Ca<sup>2+</sup> using fura-2. The fluorescence spectra of these compounds are sufficiently different to allow simultaneous measurement of pH and Ca<sup>2+</sup> both in vitro and in vivo. Moreover, the ratio of the H + - sensitive wavelengths of snarf-1 is unaffected by Ca<sup>2+</sup> , or the concomitant presence of fura-2 in cells. Although the fluorescence ratio of fura-2 is insensitive to the presence of snarf-1, it is affected by pH, as indicated above. We describe procedures to correct for this effect and to obtain calibration parameters for fura-2 and snarf-1 required to facilitate analysis of pH and Ca<sup>2+</sup> concentrations within cell populations.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages253-271
Number of pages19
Volume937
ISBN (Print)9781627030854
DOIs
StatePublished - 2013
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume937
ISSN (Print)10643745

Keywords

  • Intracellular pH
  • Snarf-1

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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    Martinez-Zaguilan, R., Tompkins, L. S., Gillies, R. J., & Lynch, R. (2013). Simultaneous analysis of intracellular pH and Ca<sup>2+</sup> from cell populations. In Methods in Molecular Biology (Vol. 937, pp. 253-271). (Methods in Molecular Biology; Vol. 937). Humana Press Inc.. https://doi.org/10.1007/978-1-62703-86-1_16