Characterization of exocytosis in electropermeabilized neutrophils by flow cytometric analysis: difference in sensitivity to calcium and guanosine-5'-[gamma-thio]triphosphate.
When rabbit neutrophils were subjected to two electrical discharges of 4.75 kV/cm, the cells became permeable to propidium iodide. Measurement of propidium iodide fluorescence using flow cytometry showed that all cells in the suspension were permeabilized. The cells remained permeable for > 20 min when the cells were stored at 0 degree C. When exocytosis was induced by Ca2+ alone, the orthogonal light scatter (a sensitive parameter for cell granularity) of the complete population changed depending on the concentration. All the cells were equally sensitive to Ca2+ and showed a similar degree of exocytosis at the same time. In the presence of a fixed concentration of Ca2+ and a variable concentration of guanosine-5'-[v-thio]triphosphate (GTP gamma S), a division of the cell population was observed in the orthogonal light scatter histogram. At low GTP gamma S concentrations, a part of the population showed complete exocytosis and a part of the population showed almost no exocytosis. With increasing GTP gamma S concentrations, the light scatter pattern of the population changed indicating that the cells were gradually sensitive to GTP gamma S. Electropermeabilized neutrophils showed an equal sensitivity to Ca2+ and a graded sensitivity to GTP gamma S. Flow cytometry is considered as an ideal tool to study such an effect on a cell-to-cell basis.
Boonen
G J
GJ
Department of Medical Biochemistry, University of Leiden, The Netherlands.
de Koster
B M
BM
van der Keur
M
M
VanSteveninck
J
J
Tanke
H J
HJ
Elferink
J G
JG
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002463:Cell Membrane Permeability / Q000502:physiology; D004594:Electrophysiology; D005089:Exocytosis / Q000187:drug effects / Q000502:physiology*; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology*; D008247:Lysosomes / Q000502:physiology / Q000648:ultrastructure; D009504:Neutrophils / Q000166:cytology* / Q000502:physiology* / Q000648:ultrastructure; D011419:Propidium; D011817:Rabbits
D011419:Propidium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D002118:Calcium