Programmed cell death or apoptosis is the most common form of eukaryotic cell death, and is a physiological suicide mechanism that preserves homeostasis, in which cell death naturally occurs during normal tissue turnover. In general, cells undergoing apoptosis display a characteristic pattern of structural changes in nucleus and cytoplasm, including rapid blebbing of plasma membrane and nuclear disintegration. The nuclear collapse is associated with extensive damage to chromatin and DNA-cleavage into oligonucleosomal length DNA fragments after activation of a calcium-dependent endogenous endonuclease. On the other hand, apoptosis is essential in many physiological processes, including maturation and effector mechanisms of the immune system, embryonic development of tissue, organs and limbs, development of the nervous system and hormone-dependent tissue remodeling. In oncology, extensive interest in apoptosis comes from the observation, that this mode of cell death is triggered by a variety of antitumor drugs, radiation and hyperthermia, and that the intrinsic propensity of tumor cells to respond by apoptosis is modulated by expression of several oncogenes and may be a prognostic marker for cancer treatment. Several methods have been described to identify apoptotic cells. Endonucleolysis is considered as the key biochemical event of apoptosis, resulting in cleavage of nuclear DNA into oligonucleosome-sized fragments. Therefore, this process is commonly used for detection of apoptosis by the typical “DNA ladder“ on agarose gels during electrophoresis. This method, however, can not provide information regarding apoptosis in individual cells nor relate cellular apoptosis to histological localization or cell differentiation. This can be done by enzymatic in situ labeling of apoptosis induced DNA strand breaks. DNA polymerase as well as terminal deoxynucleotidyl transferase (TdT) have been used for the incorporation of labeled nucleotides to DNA strand breaks in situ.
Enzyme Solution, Terminal deoxynucleotidyl transferase from calf thymus (EC 18.104.22.168), recombinant in E. coli, in storage buffer, 10×, 5 × 50 µl
Label Solution, Nucleotide mixture in reaction buffer, 1×, 6 × 500 µl
Converter-POD, Anti-fluorescein antibody, Fab fragment from sheep, conjugated with horse-radish peroxidase (POD), Ready-to-use, 5 ml
Flow Cytometry, Fluorescence Microscopy
Store between 2°C-8°C. Do not freeze.
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