Recording of intracellular Ca2+, Cl-, pH and membrane potential in cultured astrocytes using a fluorescence plate reader

A multi-well fluorescence plate reader was used to determine changes in int racellular ionic concentrations and changes in transmembrane voltage in pri mary cortical or hippocampal astrocytes. Recordings were compared to those obtained using a traditional microscope-based imaging setup that utilizes a monochromatic light source for excitation and an intensified CCD camera fo r detection. Measurement of pH(i) with the ratiometric dye BCECF provided r esolution similar to that of a microscopic approach. We also demonstrate us ing Fluo-3 that the measurement of glutamate-induced [Ca2+](i) fluctuations are comparable to recordings on the microscope-based system when 25 cells were averaged. This is expected because the plate reader averages responses from many cells. Voltage changes induced by changes in K-o(+) from 3 to 5 mM were readily resolvable with the plate reader using the potentiometric d ye bis-oxynol, and overall sensitivity was similar to that of microscopical ly-determined voltage changes. We also found that the plate reader was capa ble of resolving GABA-induced Cl-i(-) fluctuations using the Cl- -sensitive indicator MEQ. From these experiments we conclude that multi-well fluoresc ence plate readers can be used to effectively record changes in intracellul ar ion concentrations and transmembrane voltage of populations of cells aff ording time and amplitude resolution approaching that of conventional fluor escence imaging methods. In addition, plate reader-based fluorescence studi es demonstrate the added capability to rapidly screen large numbers of samp les. (C) 1999 Elsevier Science B.V. All rights reserved.