Cytometric assessment of mitochondria using fluorescent probes

Abstract

Mitochondria are most important organelles in the survival of eukaryotic aerobic cells because they are the primary producers of ATP, regulators of ion homeostasis or redox state, and producers of free radicals. The key role of mitochondria in the generation of primordial ATP for the survival and proliferation of eukaryotic cells has been proven by extensive biochemical studies. In this context, it is crucial to understand the complexity of the mitochondrial compartment and its functionality and to develop experimental tools allowing the assessment of its nature and its function and metabolism. This review covers the role of the mitochondria in the cell, focusing on its structure, the mechanism of the mitochondrial respiratory chain, the maintenance of the transmembrane potential and the production of reactive oxygen species. The main probes used for mitochondrial compartment monitoring are described. In addition, various applications using mitochondrial-specific probes are detailed to illustrate the potential of flow and image cytometry in the study of the mitochondrial compartment. This review contains a panel of tools to explore mitochondria and to help researchers design experiments, determine the approach to be employed, and interpret their results. ' 2011 International Society for Advancement of Cytometry

Arsenic Trioxide Treatment Decreases the Oxygen Consumption Rate of Tumor Cells and Radiosensitizes Solid Tumors

“…This hypothesis is supported by the fact that the number of TLT cells with depolarized mitochondria was increased after exposure to As 2 O 3 . It is important to note that changes in oxygen consumption can occur a long time before changes in membrane potential become measurable (26). Overall, these observations indicate that As 2 O 3 could act on the mitochondrial respiratory chain by enhancing intracellular ROS production mediated by decreased GSH levels.…”

“…When dispersed in this manner JC-1 fluoresces green. Consequently, mitochondrial depolarization is indicated by a decrease in the red-green fluorescence intensity ratio (26). Data acquisition was done using a FACSCalibur flow cytometer (Becton Dickinson), and data were analyzed with FlowJo software (Tree Star, Inc.).…”

The Tumor Microenvironment Represses T Cell Mitochondrial Biogenesis to Drive Intratumoral T Cell Metabolic Insufficiency and Dysfunction

“…To assay the metabolic capacity of tumor-infiltrating T cells, we measured mitochondrial function and mass by flow cytometry using MitoTracker Deep Red FM (a membrane permeable, carbocyanine-based dye for mitochondria used previously to stain mitochondrial mass; Cottet-Rousselle et al, 2011) and competency for glucose uptake using fluorescent 2-NBD-glucose (2NBDG) in T cells infiltrating implantable tumors. While Mito-Tracker Deep Red has been shown to be membrane potential sensitive in some systems, uncoupling using carbonyl cyanide m-chlorophenyl hydrazine (CCCP) showed that, using our staining protocols, MitoTracker Deep Red was highly resistant to collapse of membrane potential, especially compared to tetra-methylrhodamine ester (TMRE), a well-known membranepotential sensitive dye ( Figure S1A).…”

Lipophilic triphenylphosphonium derivatives enhance radiation-induced cell killing via inhibition of mitochondrial energy metabolism in tumor cells

“…and MitoSOX red (MSR, Thermo Fisher Scientific Co.) were used as fluorescent probes for mitochondrial membrane potential and for mitochondria-derived ROS, respectively [28]. …”