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

We have investigated, at the single cell level, intracellular Ca2+ ([C a2+](i)) modulations triggered by the high affinity receptor for IgG, Fc gamma RI, in the monocytic cell line, U937. Cells were co-loaded wi th the Ca2+-sensitive dyes, Fluo-3 and Fura-Red, by incubation with th eir acetoxymethyl (AM) esters and confocal ratio imaging was used to m onitor the [Ca2+](i) changes induced by antibody cross-linking of IgG- loaded Fc gamma RI. A single Ca2+ spike was observed in 81% of untreat ed cells whereas dibutyryl cAMP-induced differentiation into a more ma crophage cell type resulted in a sub-population of cells (44%) respond ing to receptor cross-linking with calcium oscillations. This change i n calcium signalling may explain the difference in functional response s triggered by Fc gamma RI in monocytes and macrophages. Analysis of t he Fluo-3 and Fura-Red fluorescence, after AM-ester loading, showed th at both dyes have similar photobleach rates and intracellular localiza tion allowing compensation for shifts in focal plane, dye photobleachi ng and non-uniformity of dye loading. In addition, because the binding kinetics of both dyes are equivalent, accurate temporal information c hanges. There are, however, two major problems with can be gained abou t [Ca2+] this dual indicator technique. Firstly, loading from AM ester s results in considerable variation between cells in the intracellular concentration ratio of the two dyes, making calibration difficult. Se condly, 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 con siderable amplitude distortion of the signal when the ratios are displ ayed bn a linear scale. These problems may considerably limit the appl ication of Fluo-3/Fura-Red ratiometric measurements.