Flow cytometry study of leukocyte function: analytical comparison of methods and their applicability to clinical...

Perturbations in the redox-based network of cellular regulatory mechanisms have been associated with oxidative-related diseases such as diabetes mellitus. In these situations the redox state of cellular redox systems becomes persistently shifted toward oxidation that may result in a sequence of pathophysiological events. Innate and adaptive immune responses depend on the production of reactive oxygen species and ATP synthesis, which are tightly regulated by the mitochondrial transmembrane potential. Mitochondrial hyperpolarisation is a key mechanism of T-cell life, apoptosis and autoimmunity. The NADPH oxidase of the phagocytic cells of the immune system generates reactive oxygen metabolites during the respiratory burst, but activated B cells also possess NADPH oxidase and reactive oxidants could play regulatory roles in immune function. Cellular thiol levels and the thiol reduction-oxidation process modulate the oxidative metabolism in the cells, transcriptional factor activation of gene expression, lymphocyte proliferation and death. Flow cytometry allows directly characterising and analysing several parameters and functions of intact living cells in a few seconds. Fluorescent lipophilic cations have been used for the measurement of the mitochondrial transmembrane potential. Evaluation of reactive oxygen intermediates generation in neutrophils may be obtained by use of oxidation-sensitive probes. The dye resazurin has been used to quantify mitochondrial activity since considered to act as an intermediate electron acceptor in the electron transport chain between the final reduction of oxygen and cytochrome oxidase. The fluorescence emitted by 5-chloromethyl fluorescein acetate stained cells reflects the total level of free intracellular thiol. In this review we will discuss the possible importance and consequences of evaluating these redox parameters in diabetes pathophysiology. Moreover, we will provide perspectives concerning the varieties of analytical procedures that are capable of measuring them. The advantages and disadvantages of each of these methods are critically discussed particularly in view of their clinical application.