Recording of intracellular Ca 2+, Cl −, pH and membrane potential in cultured astrocytes using a fluorescence plate reader

Abstract

A multi-well fluorescence plate reader was used to determine changes in intracellular ionic concentrations and changes in transmembrane voltage in primary 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 for detection. Measurement of pH i with the ratiometric dye BCECF provided resolution similar to that of a microscopic approach. We also demonstrate using Fluo-3 that the measurement of glutamate-induced [Ca 2+] 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 dye bis-oxynol, and overall sensitivity was similar to that of microscopically-determined voltage changes. We also found that the plate reader was capable of resolving GABA-induced Cl − i fluctuations using the Cl −-sensitive indicator MEQ. From these experiments we conclude that multi-well fluorescence plate readers can be used to effectively record changes in intracellular ion concentrations and transmembrane voltage of populations of cells affording time and amplitude resolution approaching that of conventional fluorescence imaging methods. In addition, plate reader-based fluorescence studies demonstrate the added capability to rapidly screen large numbers of samples.