Abstract
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 diagostic 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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Chilmonczyk S, Monge D (1999). Flow cytometry as a tool for assessment of the fish cellular immune response to pathogens. Fish and Shellfish Immunol 9: 319-333.
Chilmonczyk S, Monge D, Baranyiova E, Vykusova B, Svobodova Z (1998). Cellular immune response in rainbow trout (Oncorhynchus mykiss): Flow cytometric study. Acta Veterinaria 67: 207-213.
Hogan RJ, Taylor WR, Cuchens MA, Naftel JP, Clem LW, Miller NW, Chinchar VG (1999). Induction of target cell apoptosis by channel catfish cytotoxic cells. Cellular Immunology 195: 110-118.
Verbury-Van Demenade BM, Nowak B, Engelsma MY, Weyts FA (1999). Differential effects of cortisol on apoptosis and proliferation of carp B-lymphocytes from head kidney, spleen and blood. Fish and Shellfish Immunology 9: 405-415.
Gogal RM Jr, Smith BJ, Robertson JL, Smith SA, Holladay SD (1999). Tilapia (Oreochromis niloticus) dosed with azathioprine display immune effects similar to those seen in mammals, including apoptosis. Veterinary Immunology and Immunopathology, 68: 211-229.
Weyts FAA, Flik F., Rombout JHWM, Verbury-van Demenade BML (1998). Cortisol induces apoptosis in activated B cells, not in other lymphoid cells of common carp, Cyprinus carpio L. Developmental and Comparative Immunology 22: 551-562.
Weyts FAA, Flikt G., Verburh-van Demenade BML (1998). Cortisol inhibits apoptosis in carp neutrophilic granulocytes. Developmental and Comparative Immunology 22: 563-572.
Holladay SD, Smith SA, Besteman EG, Deyab ASMI, Gogal RM, Hrubec T, Robertson JL, Ahmed SA (1998). Benzo[a]pyrine-induced hypocellularity of the pronephros in tilapia (Oreochromis niloticus) is accompanied by alterations in stromal and parenchyman cells and by enhanced immune cell apoptosis. Veterinary Immunology and Immunopathology 64: 69-82.
Sweet LI, Passino-Reader DR, Meier PG, Omann GM (1998). Fish thymocyte viability, apoptosis and necrosis: in-vitro effects of organochlorine contaminants. Fish and Shellfish Immunology 8: 77-90.
Abellli L, Baldassini, MR Meschini R, Mastrolia L (1998). Apoptosis of thymocytes in developing sea bass Dicentrarchun labrax (L.). Fish and shellfish Immunology 8: 13-24.
Alford PB III, Tomasso JR, Bodine AB, Kendall C (1994). Apoptotic death of peripheral leukocytes in channel catfish: Effect of confinement-induced stress. J Aquatic Anim Health 6: 64-69.
Bishop GR, Jaso-Friedmann L, Evans DL (2000). Activation-induced programmed cell death of nonspecific cytotoxic cells and inhibition by apoptosis regulatory factors. Cell Immunol 199: 126-137.
Postlethwait J, Johnson S, Midson CN, Talbot WS, Gates M, Ballenger EW, Africa D, Andrews R, Carl T, Eison JS, Horne S, Kimmel CB, Hutchinson M, Johnson M, Rodriguez A (1994). A genetic linkage map for the zebrafish. Science 264: 699-703.
Murray JC, Buetow KH, Weber JL, Ludwigsen S, Scherpbier-Heddema T (1994). A comprehensive human linkage map with centimorgan density. Science 265: 2049-2054.
Shankey TV, Rabinovitch PS, Bagwell B, Bauer KD, Duque RE, Hedley DW, Mayall BH, Wheeless L (1993) Guidelines for implementation of clinical DNA cytometry. Cytometry 14: 472-477.
Rights and permissions
About this article
Cite this article
Evans, D.L., Bishop, G.R. & Jaso-Friedmann, L. Methods for cell cycle analysis and detection of apoptosis of teleost cells. Methods Cell Sci 22, 225–231 (2000). https://doi.org/10.1023/A:1009808528587
Issue Date:
DOI: https://doi.org/10.1023/A:1009808528587