40] Assays for detection of nitric oxide-induced apoptosis

Abstract

This chapter describes the detection of nitric oxide (NO)-induced apoptosis in mouse thymocytes as a classic cell population for the study of apoptotic processes. A method for the in situ labeling of DNA strand breaks in nuclei of cultured cells or tissue sections. The method is based on the specific binding of DNA polymerase I to the DNA strand breaks, where it replaces the damaged strand with a new DNA strand in which labeled nucleotides can be incorporated. Immunohistochemical staining of the labeled nucleotides allows the detection of apoptotic cells under the microscope or by flow cytometry. The method can be combined with immunocytochemistry for the detection of cell surface antigens for further characterization of specific cell types. A convenient method for applying NO to cells is by using S-nitroso-N-acetylpenicillamine (SNAP), a nitrosothiol that linearly generates NO for a long time without generation of significant amounts of oxygen radicals. Decomposition of SNAP is accelerated by cysteine-free thiol groups in a concentration-dependent manner. The major advantage of this technique is the detection of apoptosis at the level of single cells.