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    Home > Biochemistry News > Biotechnology News > Simultaneous Analysis of Intracellular pH and Ca2+ from Cell Populations

    Simultaneous Analysis of Intracellular pH and Ca2+ from Cell Populations

    • Last Update: 2020-11-22
    • Source: Internet
    • Author: User
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    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.
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