Abstract
The development of a sensitive, rapid and reliable nonradiometric cytotoxicity assay would significantly facilitate studies of teleost nonspecific cytotoxic cells. Such an assay would not require handling and disposal of radionuclides and it would not depend on secondary enzyme or colorimetric determinations. The requirements for this assay would consist of a one-step binding protocol which could detect early target cell membrane lesions and at very low effector:target cell ratios. In this chapter, we have developed a flow cytometry based cytotoxicity assay utilizing the uptake of propidium iodide (PI) into cells containing damaged (i.e. permeabilized) cell membranes. The basis of detection of cellular damage depended on flow discrimination of targets from effector cells by establishing 'scatter' gates from these mixtures. Teleost (catfish) anterior kidney NCC were mixed with human transformed targets (IM-9 and HL-60 cells) at effector:target cell ratios of 1, 5 and 10 and PI uptake was determined at 3 and 14 hours post-incubation. Percent specific uptake (PSU) was calculated by determining total binding capacity (TBC) (i.e. uptake of PI by cold acetone permeabilized target cells) and spontaneous binding capacity (SBC) (i.e. PI uptake by target cells incubated in media w/o effectors). This was represented by the formula PSU = [T − SBC/TBC − SBC] × 100 where T is the PI uptake of targets following addition of effector cells. Using this technique, NCC initiated target cell permeabilization as early as 30 minutes co-incubation (25:1 E:T ratio) and damaged membranes were detected in mixtures containing as few as 1:1 effector:target cell ratios. At 5:1 E:T ratios, greater than 50 PSU was determined following 14 hours co-incubation. Using these criteria, a new and sensitive cytotoxicity assay has been developed to determine NCC activity.
Similar content being viewed by others
References
McMillan DN, Secombes CJ (1997). Isolation of rainbow trout (Oncorhynchus mykiss) intestinal intraepithelial lymphocytes (IEL) and measurement of their cytotoxic activity. Fish Shellfish Immunol 7: 527-541.
Wierda WG, Mehr DS, Kim YB (1989). Comparison of fluorochrome-labeled and 51Cr-labeled targets for natural killer cytotoxicity assay. J Immunological Methods 122: 15-24.
Brunner KT, Mauel J, Cerottini JC, Chapuis B (1968). Quantitative assay of the lytic action of immune lymphoid cells on 51Cr-labeled allogeneic target cells in vitro; inhibition by isoantibody and by drugs. Immunology 14: 181-191.
Graves SS, Evans DL, Cobb D, Dawe DL (1984). Nonspecific cytotoxic cells in fish (Ictalurus punctatus) I. Optimum requirements for target cell lysis. Dev Comp Immunol 8: 293.
Evans DL, Graves SS, Cobb D, Dawe DL (1984). Nonspecific cytotoxic cells in fish (Ictalurus punctatus) II. Parameters of target cell lysis and specificity. Dev Comp Immunol 8: 303.
Evans DL, Hogan KT, Graves SS, Carlson RL, Floyd E, Dawe DL (1984). Nonspecific cytotoxic cells in fish (Ictalurus punctatus) III. Biophysical and biochemical properties affecting cytolysis. Dev Comp Immunol 8: 599.
Evans DL, Carlson RL, Graves SS, Hogan KT (1984). Nonspecific cytotoxic cells in fish (Ictalurus punctatus) IV. Target cell binding and recycling capacity. Dev Comp Immunol 8: 823.
Rights and permissions
About this article
Cite this article
Evans, D.L., Oumouna, M. & Jaso-Friedmann, L. Nonradiometric detection of cytotoxicity of teleost nonspecific cytotoxic cells. Methods Cell Sci 22, 233–237 (2000). https://doi.org/10.1023/A:1009860512658
Issue Date:
DOI: https://doi.org/10.1023/A:1009860512658