Flow cytometry in the study of mitochondrial respiratory chain disorders Karen Setterfield a,b , Andrew J. Williams c , Jennifer Donald a , David R. Thorburn d , Denise M. Kirby d , Ian Trounce e , John Christodoulou b,f, * a Department of Biological Sciences, Macquarie University, North Ryde, Sydney, NSW, Australia b Western Sydney Genetics Program, The Children’s Hospital at Westmead, Locked Bag 4001, Westmead, Sydney, NSW 2145, Australia c Department of Clinical Immunology, Royal Prince Alfred Hospital, Camperdown, Sydney, NSW, Australia d Murdoch Children’s Research Institute, Royal Children’s Hospital, Parkville, Vic., Australia e Mutation Research Centre, St. Vincent’s Hospital, Melbourne, Vic., Australia f Department of Paediatrics and Child Health, University of Sydney, Sydney, NSW, Australia Received 31 October 2001; received in revised form 1 January 2002; accepted 25 February 2002 Abstract We have developed a flow cytometric assay to measure the oxidative capacity of cultured lymphoblasts as a possible screening test for patients suspected of having a defect of the mitochondrial respiratory chain. Cells were incubated overnight in serum free media, followed by incubation with dihydroethidium with and without rotenone, and then analysed using flow cytometry to measure fluorescence. Inhibition with rotenone gave an increase in fluorescence compared to uninhibited cells. The change in fluorescence was significantly lower in four of the six patient cell lines, with a correlation between the activity of complex I and change in fluorescence. This method may be applicable to cell lines with defects in other complexes of the respiratory chain. q 2002 Elseiver Science B.V. and Mitochondria Society. All rights reserved. Keywords: Complex I; Dihydroethidium; Lymphoblasts; Electron transport chain; Respiratory chain; Dihydrorhodamine 123 1. Introduction The mitochondrion is the site of numerous meta- bolic pathways, but one of the most important is the production of energy in the form of ATP, by the process of oxidative phosphorylation (OXPHOS) (Rose, 1998). OXPHOS is composed of five enzyme complexes located in the inner mitochondrial membrane (Shoffner and Wallace, 1990). Abnormal- ities in mitochondrial respiratory chain function may be due to primary genetic defects of either the mito- chondrial or the nuclear genomes (Christodoulou, 2000). Clinically, defects of the mitochondrial respiratory chain can interfere with the function of single organs or can result in a multisystem disease (Christodoulou, 2000), and can show marked pheno- typic variability, both within and between families. Evaluation of respiratory chain disorders usually involves general investigations that are used to accu- mulate evidence of the pattern and nature of the differ- Mitochondrion 1 (2002) 437–445 1567-7249/02/$20.00 q 2002 Elseiver Science B.V. and Mitochondria Society. All rights reserved. PII: S1567-7249(02)00008-9 www.elsevier.com/locate/mito * Corresponding author. Tel.: 161-2-9845-3452; fax: 161-2- 9845-1864. E-mail address: johnc@chw.edu.au (J. Christodoulou). Abbreviations: OXPHOS, oxidative phosphorylation; mtDNA, mitochondrial DNA; DHR123, dihydrorhodamine 123; DE, dihy- droethidium; N, normal range; SDH, succinate dehydrogenase; PBS, phosphate buffered saline; COX, complex IV; r o , rho zero; O 2 2 , superoxide radical