IGG-INDUCED CA2+ OSCILLATIONS IN DIFFERENTIATED U937 CELLS - A STUDY USING LASER-SCANNING CONFOCAL MICROSCOPY AND CO-LOADED FLUO-3 AND FURA-RED FLUORESCENT-PROBES

We have investigated, at the single cell level, intracellular Ca2+ ([Ca2+](i)) modulations triggered by the high affinity receptor for IgG, Fc gamma RI, in the monocytic cell line, U937. Cells were co-loaded with the Ca2+-sensitive dyes, Fluo-3 and Fura-Red, by incubation with their acetoxymethyl (AM) esters and confocal ratio imaging was used to monitor the [Ca2+](i) changes induced by antibody cross-linking of IgG-loaded Fc gamma RI. A single Ca2+ spike was observed in 81% of untreated cells whereas dibutyryl cAMP-induced differentiation into a more macrophage cell type resulted in a sub-population of cells (44%) responding to receptor cross-linking with calcium oscillations. This change in calcium signalling may explain the difference in functional responses triggered by Fc gamma RI in monocytes and macrophages. Analysis of the Fluo-3 and Fura-Red fluorescence, after AM-ester loading, showed that both dyes have similar photobleach rates and intracellular localization allowing compensation for shifts in focal plane, dye photobleaching and non-uniformity of dye loading. In addition, because the binding kinetics of both dyes are equivalent, accurate temporal information changes. There are, however, two major problems with can be gained about [Ca2+] this dual indicator technique. Firstly, loading from AM esters results in considerable variation between cells in the intracellular concentration ratio of the two dyes, making calibration difficult. Secondly, the fluorescence ratio, Fluo-3/Fura-Red, behaves non-linearly at Ca2+ concentrations less than similar to 500 nM and comparison with Fura-2-loaded single cell Ca photometry studies suggests there is considerable amplitude distortion of the signal when the ratios are displayed bn a linear scale. These problems may considerably limit the application of Fluo-3/Fura-Red ratiometric measurements.