title: Simultaneous analysis of intracellular pH and Ca²⁺ from cell populations.
abstract: Although changes in both pH(in) and [Ca(2+)](i) 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(in) modify Ca(2+) regulatory mechanisms and changes in Ca(2+) 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(2+) binding proteins, all Ca(2+)-sensitive fluoroprobes are inherently sensitive to pH owing to competition of H(+) for the Ca(2+)-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(2+) using fura-2. The fluorescence spectra of these compounds are sufficiently different to allow simultaneous measurement of pH and Ca(2+) both in vitro and in vivo. Moreover, the ratio of the H(+)-sensitive wavelengths of snarf-1 is unaffected by Ca(2+), 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(2+) concentrations within cell populations.
authors.affiliation: Department of Physiology, Texas Tech University Health Sciences Center, Lubbock, TX, USA.
authors.forename: Raul
authors.initials: R
authors.lastname: Martinez-Zaguilan
authors.forename: Linda S
authors.initials: LS
authors.lastname: Tompkins
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Gillies
authors.forename: Ronald M
authors.initials: RM
authors.lastname: Lynch
mesh_terms: D001578:Benzopyrans / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009284:Naphthols / Q000378:metabolism; D012235:Rhodamines / Q000378:metabolism
chemical_list: D001578:Benzopyrans; D009284:Naphthols; D012235:Rhodamines; C091666:seminaphthorhodaminefluoride; D002118:Calcium; D016257:Fura-2