Abstract
Mitochondrial dysfunction is associated with perturbations in the cellular oxidative status, changes in energy production and metabolic rate, and the onset of pathological processes. Classic methods of assessing mitochondrial dysfunction rely on indirect measures, such as evaluating mitochondrial DNA copy numbers, or direct but more costly and skilled techniques, such as electron microscopy. The protocol presented here was recently implemented to evaluate mitochondrial dysfunction in response to insecticide exposure in Drosophila melanogaster larvae, and it relies on the use of a previously established MitoTimer mutant strain. MitoTimer is a genetically engineered mitochondrial protein that shows green fluorescence when newly synthetized, irreversibly turning into red as mitochondria age. The protocol described here allows for the easy and direct assessment of shifts in mitochondrial turnover, with tissue-specific accuracy. This protocol can be adapted to assess changes in mitochondrial turnover in response to drugs, rearing conditions, and/or mutations in larva, pupa, or adult fruit flies.