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Microbiology 147 (2001), 3335-3343
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Microbiology (2001), 147, 3335-3343.
© 2001 Society for General Microbiology

Bioenergetics and Transport

Assessment of mitochondrial membrane potential in yeast cell populations by flow cytometry

Paula Ludovico1, Filipe Sansonetty2 and Manuela Côrte-Real1

Centro de Ciências do Ambiente – Departamento de Biologia, Universidade do Minho, Campus de Gualtar, 4719-057 Braga Codex, Portugal1
Laboratório de Citometria, Instituto de Patologia e Imunologia Molecular da Universidade do Porto, Porto, Portugal2

Author for correspondence: Manuela Côrte-Real. Tel: +351 253 604314. Fax: +351 253 678980. e-mail: mcortereal{at}bio.uminho.pt

In yeast the use of rhodamine 123 (Rh123) has been restricted to the evaluation of mitochondrial respiratory function including the discrimination between respiratory-competent and -deficient cells. This study describes the optimization and validation of a low-concentration Rh123 staining protocol for the flow-cytometric assessment of mitochondrial membrane potential ({Delta}{Psi}m) changes in whole yeast cells. The optimized protocol was validated by the use of compounds that specifically affect mitochondrial energetics. Epifluorescence microscopy was used to monitor Rh123 distribution within the cell. Incubation of yeast cell suspensions with Rh123 (50 nM, 10 min) gave minimal non-specific binding and cytotoxicity of the dye. The ratio (R) between the green fluorescence and forward scatter (both measured as log values) was used to measure {Delta}{Psi}m with only little dependence on cell ‘volume’ and mitochondrial concentration. Cells treated with mitochondrial membrane de- or hyper-polarizing agents displayed a decrease and an increase of R values respectively, indicating that changes of the Rh123 distribution in cells indicate variations in the {Delta}{Psi}m. Live and dead cells also displayed significantly different R values. The method described here allows assessment of {Delta}{Psi}m changes in whole yeast cells in response to a given drug. Moreover, the relationship between drug effects and disorders of mitochondrial energetics might be addressed.

Keywords: mitochondrial functionality, flow cytometry, rhodamine 123, whole yeast cells

Abbreviations: EFM, epifluorescence microscopy; FCM, flow cytometric microscopy; FS, forward scatter; R, ratio; Rh123, rhodamine 123; SS, side scatter




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