Abstract
A series of optimized protocols to isolate vacuoles from both yeast and plant cells, and to characterize the purified organelles at a functional and structural level, are described. For this purpose, we took advantage of the combined use of cell fractionation techniques with different fluorescence-based approaches namely flow cytometry, fluorescence microscopy and spectrofluorimetry. These protocols altogether constitute valuable tools for the study of vacuole structure and function, as well as for the high-throughput screening of drug libraries to identify new molecules that target the vacuole.
Keywords:
Flow cytometry; Fluorescence microscopy; Isolated vacuoles; Plant cell; Yeast.
MeSH terms
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Acridine Orange / analysis
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Aniline Compounds / analysis
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Barbiturates / analysis
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Calcium / analysis
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Calcium / metabolism
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Cell Fractionation / methods*
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Flow Cytometry / methods*
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Fluorescent Dyes / analysis
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Isoxazoles / analysis
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Microscopy, Fluorescence / methods*
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Neutral Red / analysis
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Pyridinium Compounds / analysis
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Quaternary Ammonium Compounds / analysis
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Staining and Labeling / methods
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Vacuolar Proton-Translocating ATPases / analysis
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Vacuolar Proton-Translocating ATPases / metabolism
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Vacuoles / chemistry
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Vacuoles / enzymology
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Vacuoles / metabolism*
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Vacuoles / ultrastructure*
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Vitis / chemistry
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Vitis / cytology*
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Vitis / enzymology
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Vitis / metabolism
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Xanthenes / analysis
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Yeasts / chemistry
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Yeasts / cytology*
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Yeasts / enzymology
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Yeasts / metabolism
Substances
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Aniline Compounds
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Barbiturates
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FM1 43
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Fluo 4
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Fluorescent Dyes
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Isoxazoles
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Pyridinium Compounds
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Quaternary Ammonium Compounds
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Xanthenes
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bis(1,3-dibutylbarbiturate)trimethine oxonol
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Neutral Red
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Vacuolar Proton-Translocating ATPases
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Acridine Orange
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Calcium