Uptake of reconstituted Na,K-ATPase vesicles by isolated lymphocytes measured by FACS, confocal microscopy and spectrofluorometry |
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Authors: | Beatrice M Anner B Volet |
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Institution: | (1) Department of Pharmacology, Geneva University Medical School, CH-1211 Geneva 4, Switzerland |
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Abstract: | Na,K-ATPase (EC, 3.6.1.37, Na,K-ATPase) is a fundamental vital membrane transport and receptor system which, after biosynthesis,
is exported to the plasma membrane in inside-out vesicles. Na,K-ATPase can be extracted form the natural membrane and inserted
into artificially formed phosphatidylcholine vesicles (liposomes). The ultrastructure of the reconstituted vesicles has been
fully described. In the present work, the Na,K-ATPase-vesicles were labeled with fluorescent tracers either in their water
or membrane phase, incubated with freshly isolated human lymphocytes, and the resulting cellular fluorescence measured with
fluorescence activated cell sorting (FACS), confocal microscopy and spectrofluorometry. The FACS data show that all lymphocytes
take up Na,K-ATPase-vesicles in a dose-and temperature-dependent fashion. Three-dimensional analysis of the fluorescence by
confocal microscopy reveals that the fluorescence is contained within the cells. Quantitative determination by spectrofluorometry
indicates that depending on the vesicle/cell ratio, a single lymphocyte takes up 650 to 36,500 vesicles within 30 min at 37°C
together with up to about 200,000 renal Na,K-ATPase molecules. |
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Keywords: | Reconstituted Na K-ATPase-vesicles lymphocytes uptake FACS confocal microscopy spectrofluorometry quantitative analysis |
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