Indexed on: 30 Mar '10Published on: 30 Mar '10Published in: Methods
All mitochondrial DNA (mtDNA)-encoded genes are involved in mitochondrial electron transport and ATP production. Alterations of mtDNA due to dysfunctional mitochondrial DNA polymerase gamma (POLG) induce loss of mitochondrial oxidative phosphorylation (OXPHOS) and mitochondrial ATP generation. Total intracellular ATP is generated by two energetic pathways, glycolysis and mitochondrial OXPHOS. Decreased ATP generation from mitochondria due to mitochondrial dysfunction induces compensatory upregulation of cytoplasmic glycolysis process, thus increasing the contribution of glycolysis to the total cellular ATP generation. Decreased mitochondrial respiration and ATP generation with concomitant enhanced glycolysis is associated with mitochondrial disease and cancer. This chapter introduces a novel assay using a pharmacological profiling strategy in combination with a Seahorse XF24 instrument, which quantifies mitochondrial oxygen consumption rate and extracellular acidification rate for the measurement of OXPHOS and glycolysis, respectively. This assay combined with an analysis of steady-state ATP levels was used to study the bioenergetics of cells depleted of mtDNA (rho0 cells).