Methods for cell cycle analysis and detection of apoptosis of teleost cells.
Flow cytometric techniques have not been previously used on a routine basis to study teleost cell growth and development. In the present chapter, flow instrumentation and cell preparation protocols are given in order to provide evaluation criteria characteristic of different phases of the cell cycle. Flow cytometry is used as an analytical and diagnostic tool to measure DNA ploidy as well as to measure alterations in cell cycle profiles characteristic of random DNA fragmentation (necrosis) compared to patterned DNA cleavage (apoptosis). The types of information obtained by flow analysis include the visualization of cell subpopulations with differing DNA content. For each identified nuclei subpopulation, the parameters of population size, fractions of nuclei in each phase of the cell cycle and computation of DNA ratios can be discerned. Data are presented of ex vivo prepared teleost nonspecific cytotoxic cells (NCC) at resting phase compared to NCC undergoing DNA hypoploid changes characteristic of apoptosis. These cells are compared with a teleost tissue cultured cell line maintained under optimum cell growth conditions versus cells undergoing necrotic cellular pathology. Finally, the requirements for optimum flow analysis are described. Techniques including gating strategies, voltage and gain settings, discrimination options and data collection and interpretation are provided.
Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA. devans@calc.vet.uga.edu
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Evans
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Bishop
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Jaso-Friedmann
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002397:Catfishes; D002453:Cell Cycle / Q000502:physiology*; D002460:Cell Line; D053938:DNA Fragmentation; D004171:Diploidy; D005434:Flow Cytometry / Q000379:methods*; D066298:In Vitro Techniques; D009336:Necrosis