Techniques for measuring and manipulating free Ca2+ in the cytosol and organelles of neutrophils.

Ca(2+) signalling in neutrophils is important for triggering and coordinating the behaviour of neutrophils. Fluorescent probes for cytosolic free Ca(2+) concentration, e.g., fura2 and fluo3, have been widely used in neutrophils. These probes can be used to monitor Ca(2+) in the cytosol, the nucleus, near the plasma membrane and theoretically within Ca(2+) storage organelles. The longer wavelength indicators, e.g., fluo3 and calcium green, can be used confocally to monitor subcellular Ca(2+) changes in the cytosol of neutrophils and in the nucleus. Confocal techniques also permit "impossible views" imaging of Ca(2+) and newer scanning techniques promise very fast temporal resolution. Techniques using chlortetracycline (CTC) and DiOC(6)(3) are also described for monitoring the position of Ca(2+) storage sites in neutrophils and for manipulating their activity. Thus, in this review, a spectrum of new (and older) optical techniques are presented which are useful for measuring, monitoring and manipulating cytosolic free Ca(2+) concentration and Ca(2+) storage in neutrophils. With these techniques, it is hoped that more insight will be gained into both the mechanism of and the consequences of Ca(2+) signalling in neutrophils.

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.