Use of fluorescent dyes for measurement and localization of organelles associated with Ca2+ store release in human neutrophils

Fura-2 and its lipid analogue, FFP-18, were used to measure changes in cytosolic free Ca2+ concentration within human neutrophils. Whereas fura-2 was employed to monitor cytosolic Ca2+ increases throughout the cytosol, FFP-18 was used to monitor Ca2+ changes only near the membrane. This latter probe was incorporated into the plasma membrane as its acetoxymethyl ester (FFP-18-AM) but as de-esterification was catalysed by cytosolic esterases, the Ca(2+)-sensing probe (FFP-18 acid) accumulated on the inner face of membrane. The fluorescence of esterified probe on the extracellularly facing membrane leaflet was quenched by the membrane-impermeant ion Ni2+. Under these conditions, near membrane Ca2+ changes which resulted from the release of Ca2+ from intracellular stores was possible by conventional ratio fluorescence measurement of FFP-18. From the timing of arrival of Ca2+ at the plasma membrane, it was proposed that there were two Ca2+ storage sites, liberated by different stimuli, one close to the plasma membrane and the other more distant. In order to discover whether organelles within the neutrophil had distributions which correlate with the Ca2+ release sites, fluorescent dyes for structures within the cytosol were employed. We have previously shown that the location of the intracellular membrane stain, DiOC6 (3) corresponds to the distant Ca2+ release site. Here a second stain, BODIPY-C5 ceramide, has also been used and is shown to stain a peripheral region of the neutrophil, in a similar pattern to the near membrane Ca2+ storage site. These data therefore raise the question of whether these stains mark the organelles in neutrophils which are the two Ca2+ storage and release sites.