Title

Optimizing a Method for Determining the Mitochondrial Membrane Potential of C. elegans

Presenter Information

Samantha Gaudette

Document Type

Oral Presentation

Location

SURC Ballroom C/D

Start Date

16-5-2013

End Date

16-5-2013

Abstract

C. elegans are microscopic worms used in genetic and biochemical studies. In this study four different parameters of a fluorescence assay for determining mitochondrial membrane potential were examined. The fluorescent dye used in the assay works in dynamic equilibrium with the worm mitochondria, which is dependent on the worm and dye concentration. To best observe the effects of the dye we tested different solvents, dye concentrations, worm concentrations, and data analysis methods. The effectiveness of two solvents, dimethylsulfoxide and ethanol, was examined. Comparisons of the fluorescence emission spectra and analysis of the λ Max (emission spectrum peak) established that 80 nM dye in ethanol provided the optimal conditions. The ideal quantity of worms was determined by comparing the intensity in counts per unit (CPU) of the emission spectra of samples and it was found that approximately 900 worms provided the most repeatable results. Five different forms of statistical data analysis for smoothing the emission spectra data were compared. Observing no significant difference between smoothed data and raw data, no data smoothing was applied. We were able to measure mitochondrial membrane potential in C. elegans by noting a 10 nm shift in λ Max between samples containing dye only and samples containing worms with dye. A decrease in the λ Max shift was observed in the presence of the chemical mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), suggesting we are measuring membrane potential in the worms. This method will be used to investigate the effects of high fat diets on mitochondrial function.

Poster Number

39

Faculty Mentor(s)

Carin Thomas, Lucinda Carnell

Additional Mentoring Department

Chemistry

Additional Mentoring Department

Biological Sciences

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May 16th, 8:20 AM May 16th, 10:50 AM

Optimizing a Method for Determining the Mitochondrial Membrane Potential of C. elegans

SURC Ballroom C/D

C. elegans are microscopic worms used in genetic and biochemical studies. In this study four different parameters of a fluorescence assay for determining mitochondrial membrane potential were examined. The fluorescent dye used in the assay works in dynamic equilibrium with the worm mitochondria, which is dependent on the worm and dye concentration. To best observe the effects of the dye we tested different solvents, dye concentrations, worm concentrations, and data analysis methods. The effectiveness of two solvents, dimethylsulfoxide and ethanol, was examined. Comparisons of the fluorescence emission spectra and analysis of the λ Max (emission spectrum peak) established that 80 nM dye in ethanol provided the optimal conditions. The ideal quantity of worms was determined by comparing the intensity in counts per unit (CPU) of the emission spectra of samples and it was found that approximately 900 worms provided the most repeatable results. Five different forms of statistical data analysis for smoothing the emission spectra data were compared. Observing no significant difference between smoothed data and raw data, no data smoothing was applied. We were able to measure mitochondrial membrane potential in C. elegans by noting a 10 nm shift in λ Max between samples containing dye only and samples containing worms with dye. A decrease in the λ Max shift was observed in the presence of the chemical mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), suggesting we are measuring membrane potential in the worms. This method will be used to investigate the effects of high fat diets on mitochondrial function.