USE OF FLUORESCENT DYES FOR MEASUREMENT AND LOCALIZATION OF ORGANELLES ASSOCIATED WITH CA2+STORE RELEASE IN HUMAN NEUTROPHILS
✍ Scribed by ERYL V. DAVIES; HELEN BLANCHFIELD; MAURICE B. HALLETT
- Publisher
- Elsevier Science
- Year
- 1997
- Tongue
- English
- Weight
- 213 KB
- Volume
- 21
- Category
- Article
- ISSN
- 1065-6995
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✦ Synopsis
Fura-2 and its lipid analogue, FFP-18, were used to measure changes in cytosolic free Ca 2+ concentration within human neutrophils. Whereas fura-2 was employed to monitor cytosolic Ca 2+ increases throughout the cytosol, FFP-18 was used to monitor Ca 2+ 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 membraneimpermeant ion Ni 2+ . Under these conditions, near membrane Ca 2+ changes which resulted from the release of Ca 2+ from intracellular stores was possible by conventional ratio fluorescence measurement of FFP-18. From the timing of arrival of Ca 2+ at the plasma membrane, it was proposed that there were two Ca 2+ 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 Ca 2+ release sites, fluorescent dyes for structures within the cytosol were employed. We have previously shown that the location of the intracellular membrane stain, DiOC 6 (3) corresponds to the distant Ca 2+ release site. Here a second stain, BODIPY-C 5 ceramide, has also been used and is shown to stain a peripheral region of the neutrophil, in a similar pattern to the near membrane Ca 2+ storage site. These data therefore raise the question of whether these stains mark the organelles in neutrophils which are the two Ca 2+ storage and release sites.