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Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labeling or the TUNEL assay is an important technique in the assessment of DNA damage. Semen samples are routinely assessed microscopically to assess their fertilization ability. In addition to routine semen analysis, the use of the TUNEL assay can provide information on the level of DNA damage present within a sample. This chapter provides a practical walk-through guide aimed at directing a researcher or a clinical facility interested in setting up and using TUNEL and flow cytometry or fluorescence microscopy for sperm DNA analysis.

Terminal dUTP nick end labeling (TUNEL) is an invaluable technique used in the study of late-stage apoptosis. The technique is based upon detection of fragmented DNA, a well-recognized characteristic of apoptosis, usually with fluorescent markers. Here, we describe the TUNEL technique (1) employing two different detection techniques, fluorescence microscopy and fluorescence-activated cell sorting (2) which can be applied to the analysis of apoptosis in retinal tissues or retinal cell cultures, respectively.

Apoptosis is a natural process occurring during the development of central nervous system resulting in the elimination of neurons that may have formed faulty synapses. Apoptosis can also be triggered by deprivation of growth factors or by exposure to a variety of endogenous or exogenous compounds. Several methods exist to assess apoptosis in neuronal cells. The choice of a particular method for apoptosis detection is dependent on the cell system, the nature of the toxin or toxicant, the type of information being sought, and, finally, on technical limitations. In this chapter, we describe techniques to evaluate apoptosis in primary neuronal cell cultures based on the evaluation of three criteria: cell morphology, biochemical changes, and DNA degradation. To draw correct conclusions regarding the mode of cell death, a combination of some of the methods mentioned above should be used.

Recently, the concept of apoptotic cell elimination was expanded and programed cell death is no longer viewed as an individual cellular event. The complete description of the apoptotic process now includes two phases: the self-driven cell disassembly and the externally-controlled elimination of apoptotic cell corpses by phagocytizing cells. The second, phagocytic phase is essential, highly conserved, and is even more important than the internal phase of cell disassembly. This is because it ensures the complete degradation of the dying cell's DNA, preventing the release of pathological, viral and tumor DNA, and self-immunization. In different cells and species from mammals to flies, a single conserved enzyme--DNase II is responsible for the elimination of cellular DNA in the second "mopping up" phase of apoptosis. Here, we present an assay for the selective detection of the phagocytic phase of apoptosis. The technology capitalizes on the fact that phagocytic DNase II produces identifiable signature DNA breaks, which can be labeled by vaccinia topoisomerase. The assay permits labeling of the previously underestimated phase of apoptotic execution and is a useful tool in the apoptosis detection arsenal.

Apoptosis is an important process both in normal biology and in various pathologies and disease states. Apoptosis in tissue or cells can be detected in a number of ways. In tissue sections, electron microscopy can identify apoptosis by cellular and nuclear morphology, and in live cells, changes in the membrane and membrane permeability allow apoptosis and necrosis to be observed. Histologically, apoptosis is best detected using the partial DNA degradation that is present in apoptotic cell nuclei. Terminal transferase-mediated UTP nick end-labeling (TUNEL) has been used successfully for detection of DNA degradation in paraffin-embedded tissue sections and can be combined with immunohistochemistry if desired to allow more precise identification of apoptotic cells.

Apoptosis is a distinct form of cell demise. Originally defined by cellular morphology, apoptosis can now be characterized at the molecular, biochemical, and cellular levels. The detection of apoptosis has become more important, not only in the interests of scientific investigation, but also because of its significance in clinical practice. For example, since apoptosis has been implicated in a variety of devastating diseases such as cancer, therapies targeting apoptosis are being developed. To evaluate the effectiveness of the treatment, one would have to assess the apoptotic response before, during, and after the therapy. For the typical apoptosis, a set of characteristics in cell structure and biochemistry has been well defined. In combination, these provide the basis for apoptosis detection in a given setting. The methodology for analyzing these characteristics is as diverse as the research subjects. Several books devoted to the methodology of apoptosis analysis have recently been published (1–3). Readers may find detailed experimental protocols in these books. This chapter provides an overview of the basic approaches used in analyzing apoptosis, the principle and the basic methodology, in order to provide a quick guide for readers to use when deciding which methods are available and appropriate for their own study. We start with the determination of cell viability and the morphology of dying cells. We then discuss approaches for examining apoptotic changes on the cell membrane, in the cytosol, and in the nucleus. We also summarize some of the common sources of reagents for apoptosis research.

DNA damage caused by endogenous and exogenous agents represents a serious survival challenge for cells. The cell-cycle checkpoints and DNA repair mechanisms are activated to allow more time for effective repair. However, if these mechanisms fail or the damage is irreparable, then apoptosis is induced to eliminate cells carrying DNA lesions and preserve genomic integrity. This chapter discusses how DNA damage-dependent apoptosis is induced and executed.

Cell death by apoptosis is now recognized widely as an important constituent of cell turnover and disease pathology. Characterized by the cleavage of DNA into oligonucleosome-sized DNA fragments, end-labeling of fragmented DNA often is used as an in situ histological marker of apoptosis. The judicious and appropriate use of this technique therefore provides us with an important tool for assessing cell kinetics. Protocols for both terminal transferase-mediated UTP nick end-labeling, so-called TUNEL, and the combination of TUNEL with immunohistochemical staining are presented here, along with a discussion of its significance and interpretation.

Classical apoptotic cell death can be defined by certain morphological and biochemical characteristics that distinguish it from other forms of cell death. One such feature, which is a hallmark of apoptosis, is DNA fragmentation. In dying cells, DNA is cleaved by an endonuclease that fragments the chromatin into nucleosomal units, which are multiples of about 180-bp oligomers and appear as a DNA ladder when run on an agarose gel. Here, we present commonly used methods such as conventional agarose gel electrophoresis to analyze fragmented nuclei in cells. The various methods used are dependent on the extent of fragmentation or the amount of fragmented nuclei in a sample. Determining whether a cell exhibits DNA fragmentation can provide information about the type of cell death occurring and the pathways activated in the dying cell.

Developing sympathetic neurons, which depend on nerve growth factor for survival, are one of the best studied in vitro models of neuronal apoptosis and have been extensively used for cellular and molecular studies of the neuronal death pathway. Important apoptotic events after nerve growth factor withdrawal include the release of proapoptotic proteins, such as cytochrome c, from the mitochondria and the activation of caspases, followed by nuclear DNA fragmentation and chromatin condensation. In this chapter, we describe immunocytochemical techniques for studying apoptotic DNA fragmentation, changes in nuclear morphology, and mitochondrial cytochrome c release at the single cell level using sympathetic neurons cultured on glass coverslips.

A double-labeling assay for detecting apoptotic cells, using the terminal deoxy-nucleotidyl transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) assay, and antigens of interest, using immunofluorescence, is described. The assay has been used successfully on fixed, cultured cells and formalin-fixed paraffin-embedded tissue sections. This in situ detection system can be used to correlate apoptosis with specific antigen expression to identify factors involved in the induction and execution of the apoptotic pathway of cell death in a wide variety of experimental systems.

Apoptosis is a series of controlled sequential events resulting in cell death. This complements proliferation in the maintenance of tissue homeostasis. The process is regulated to give a "shrinking cell" with a charactertstic appearance. Apoptotic cells undergo compaction of nuclear chromatin and cytoplasmic condensation followed by budding of the cell to form membrane bound apoptotic bodies (1, 2). These contain varying proportions of cellular organelles and nuclear material and are rapidly phagocytosed by surrounding cells. Cell loss, therefore, is achieved without the induction of inflammation. The discovery that certain proto-oncogenes (3), e.g., c-myc and bcl-2 and the tumor suppressor gene p53 are implicated in the control of apoptosis has focused attention on both the role of apoptosis in tumorogenesis and as a possible pathway to which cancer therapeutic regimens could be directed (4). The latter point is clinically relevant in that radiotherapy, hormone therapy, and a broad spectrum of chemotherapeutic agents all induce apoptotic death in target cells. Therefore, tumor resistance to these agents may well be associated with blockades or lesions in the apoptotic pathway.

The diphenylamine assay is a very useful tool in measuring apoptosis by determining the percentage of fragmentation of known amounts of DNA into oligosomal-sized fragments. Another advantage of the diphenylamine assay is that apoptotic DNA fragmentation can be analyzed in both adherent and shed cells following treatment or experimentation. Data obtained from the experiment are expressed as a percentage relative to uninduced or untreated controls. This assay was first described by Dische (1,2) in the 1930s and later modified by Burton (3) in the mid-1950s. These modifications have resulted in an enhanced sensitivity of up to five times by adding sulfuric acid and acetaldehyde, and by allowing the colorimetric reaction to develop overnight at room temperature. These changes have also resulted in a reduction of the interference from other substances that were an initial drawback with the originally described method, further enhancing the sensitivity of the assay. The diphenylamine reaction takes advantage of the bonds between purines and deoxyribose, which are very labile. Once these bonds are broken, inorganic phosphates are liberated from the DNA and provide the substrate, which is measured by the reaction. The overall preparation time is approximately 3 h for 30 samples with incubation occurring overnight for 12 to 16 h. Reading of the results takes approximately 1 min per sample. This assay has been used to detect apoptotic fragmentation by others, and more recently by our group to evaluate cisplatin and Taxol induced fragmentation in ovarian carcinoma cell lines (4).