PMID: 3755432Jul 25, 1986Paper

Simultaneous measurement of stimulus-induced changes in cytoplasmic Ca2+ and in membrane potential of human neutrophils.

The Journal of Biological Chemistry
K G LazzariE R Simons

Abstract

The activation of human neutrophils by chemotactic peptides evokes a rapid change in membrane potential and an increase in cytoplasmic Ca2+ levels. These events are followed up to a minute later by detectable levels of microbicidal agents formed by the oxidative burst. Except for the latter, the sequence of events has remained unclear. We report here that a new fluorescent Ca2+ indicator developed by R. Tsien, Indo-1, has allowed us to resolve the temporal relationship between the rapid and transient cytoplasmic Ca2+ rise and the membrane potential change and to do so on very small samples by using a fluorescence-activated cell sorter. We have adapted a FACS 440 for simultaneous single cell membrane depolarization and cytoplasmic [Ca2+] detection in human neutrophils upon stimulation with formyl-methionyl-leucyl-phenylalanine (fMLP). A membrane potential probe, dipentyloxacarbocyanine, allows us to determine that the membrane potential change is fMLP dose-dependent and apparently biphasic. The depolarization is maximal 40 s after stimulation. In contrast, cytosolic [Ca2+], while fMLP-dose dependent, is maximal at 10 s and already decreasing rapidly when the cell has reached its lowest potential. It can be measured with Indo-1 w...Continue Reading

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