Nonradiometric detection of cytotoxicity of teleost nonspecific cytotoxic cells.
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] x 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.
Evans
D L
DL
Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA. devans@calc.vet.uga.edu
Oumouna
M
M
Jaso-Friedmann
L
L
D000818:Animals; D002397:Catfishes; D002463:Cell Membrane Permeability / Q000502:physiology*; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D066298:In Vitro Techniques; D007668:Kidney / Q000166:cytology; D008297:Male; D011419:Propidium; D018675:Toxicity Tests / Q000379:methods*
D011419:Propidium