Fluorescence measurement of cytosolic pH in cultured rodent astrocytes

This chapter presents various methods used in the laboratories for the (1) isolation and primary culture of rodent astroglia, (2) measurement of pH in the populations of glial cells using a pH-sensitive fluorescent probe, (3) calibration of the fluorescence signal, and (4) manipulation of pH_i as means of investigating the proton transport mechanisms present in the plasma membrane of these cells. These methods can easily be adapted for use with other types of cultured or freshly isolated cells. Such techniques allow micro spectrofluorimetry and fluorescence imaging of pH at the single-cell and subcellular levels. With appropriate choices of indicator dyes and excitation and emission wavelengths, a simultaneous monitoring of multiple ion species is feasible. It is now possible to monitor both membrane currents and pH_i by simultaneous patch-clamp recording and microspectrofluorimetry. These developments will undoubtedly lead to a better understanding of the physiological and pathological roles of H transporters in glia and other cell types.