24] Flow cytometry technique for assessing effects of N-acetylcysteine on apoptosis and cell viability of human immunodeficiency virus-infected lymphocytes

Abstract

This chapter discusses the flow cytometry technique for assessing the effects of N-acetylcysteine on apoptosis and cell viability of human immunodeficiency virus (HIV)-infected lymphocytes. There is a severe oxidative stress affecting HIV-infected individuals with a high consumption of antioxidants. Glutathione (GSH) is a major constituent of the cellular and plasmatic redox regulation system. It is present in most cells at a relatively high concentration. A decrease in GSH has been correlated to a molecular process leading to cell damage and cell death. Glutathione deficiency could facilitate HIV replication and the global depression of immune function. Therefore, it is interesting to study the restoration of global cellular viability by repleting redox pathways with N-acetylcysteine (NAC), which is active as a precursor of GSH. A fluorescence microscopy technique is adopted to flow cytometry to evaluate cell viability, using ethidium bromide (EB) and acridine orange (AO). Ethidium bromide enters dead cells in a passive manner. When bound to DNA, it emits a red-orange fluorescence under 488-nm laser excitation. Fluorescence-activated cell sorting (FACS) phenotyping of peripheral blood lymphocytes during antioxidant treatment of HIV-infected individuals suggests that the antioxidant defense system is essential for the regulation of T cell subsets. The chapter also discusses the expression of transglutaminase, an enzyme that is considered an effector element of gene regulation in cell death.