ABSTRACT
Interest in the investigation of mitochondrial dysfunction has seen a resurgence over recent years due to the implication of such dysfunction in both drug-induced toxicity and a variety of disease states. Here we describe a methodology to assist in such investigations whereby the oxygen consumption of isolated mitochondria is assessed in a high-throughput fashion using a phosphorescent oxygen-sensitive probe , standard microtiter plates, and plate reader detection. The protocols provided describe the required isolation procedures, initial assay optimization, and subsequent compound screening. Typical data is also provided illustrating the expected activity levels as well as recommended plate maps and data analysis approaches.
Subject(s)
Biological Assay/methods , High-Throughput Screening Assays/methods , Mitochondria/metabolism , Oxygen Consumption/drug effects , Polarography/methods , Animals , Biological Assay/instrumentation , Cell Respiration/drug effects , High-Throughput Screening Assays/instrumentation , Liver/cytology , Mitochondria/drug effects , Myocytes, Cardiac/cytology , Oxygen/metabolism , Polarography/instrumentation , Rats , Rats, Sprague-Dawley , Toxicity Tests/instrumentation , Toxicity Tests/methodsABSTRACT
Interest in the investigation of mitochondrial dysfunction has seen a resurgence over recent years due to the implication of such dysfunction in both drug-induced toxicity and a variety of disease states. Here, we describe a methodology to assist in such investigations whereby the oxygen consumption of isolated mitochondria is assessed in a high-throughput fashion using a phosphorescent oxygen-sensitive probe, standard microtitre plates, and plate reader detection. The protocols provided describe the required isolation procedures, initial assay optimization, and subsequent compound screening. Typical data is also provided illustrating the expected activity levels as well as recommended plate maps and data analysis approaches.
