QUANTITATIVE ASSAY BY FLOW-CYTOMETRY OF THE MITOCHONDRIAL-MEMBRANE POTENTIAL IN INTACT-CELLS

Mitochondrial membrane potential, in situ, is an important indicator o f mitochondrial function and dysfunction. Because of recent interest i n the role of mitochondria in signaling, cell injury and cell death, t here is a need for a convenient, sensitive and accurate method for the measurement of the mitochondrial membrane potential, Delta psi(m), in situ, in a heterogeneous cell population. We have adapted a flow cyto metry method for the quantitative measurement of Delta Psi(m) which ut ilizes the lipophilic, cationic, fluorescent probe 3,3'-dihexyloxacarb ocyanine iodide (DiOC(6)(3)). We developed a new protocol in which cel ls are equilibrated with very low dye concentrations (< 1 nM). Only un der these condition, the cell fluorescence appears to be correlated wi th the magnitude of Delta Psi(m), as evident from the sensitivity of t he fluorescence to low concentrations of uncouplers, ionophores and in hibitors of the mitochondrial proton pumps. The magnitude of the plasm a membrane potential, Delta Psi(p), also affects cell fluorescence, an d a procedure that corrects for this effect is outlined. This method o ffers a distinct advantage over existing methods for estimation of Del ta psi(m) by flow cytometry. 0167-4889/98/$ - see front matter (C) 199 8 Elsevier Science B.V. All rights reserved.