Flow cytometry-based assay for determination of teleost cytotoxic cell lysis of target cells.
BACKGROUND
The nonradiometric assays previously developed to detect cellular cytotoxic activity have been hindered by many difficulties. Among the problems are the requirement for expensive commercial kits and the use of techniques that produce high background noise and decreased sensitivity. In addition, these assays did not account for bidirectional apoptosis (activation-induced cell death [AICD]). Most attempts to derive cytometry-based cytotoxicity assays have been unsuccessful because individual effectors and targets could not be identified (i.e., "separated") using gating techniques.


METHODS
In the present study, teleost nonspecific cytotoxic (NCC) and mammalian target cells were each sufficiently different in size to identify them by flow cytometry (FCM). Using appropriate gating and discriminator techniques, these two cell populations were differentiated based on scatter properties and propidium iodide (PI) binding. Total capacity for PI binding was obtained by permeabilization of the targets with ice-cold acetone. Spontaneous PI binding was relatively low. This technique detected cytotoxicity at effector-to-target ratios (E:T) of 1:1 and after only 30 min cocultivation.


RESULTS
Tilapia NCC from peripheral blood kill human transformed target cells by necrosis and apoptosis as identified by PI binding. Maximum killing of HL-60 targets (approximately 100%) occurred by 180 min cocultivation. For the same time, the killing of IM-9 did not exceed 60%. Almost 90% of IM-9 targets are lysed following 14 h of cocultivation. The maximum killing of both HL-60 and IM-9 targets was observed at a 25:1 E:T ratio after 14 h. Comparisons of the chromium(>51) release assay with flow detection of cytotoxicity revealed that FCM detected 55% lysis of the target cells compared with 2% cytotoxicity by chromium release, after a cocultivation time of 240 min.


CONCLUSIONS
FCM detection of (teleost) NCC lysis of target cells using PI uptake is more sensitive than standard chromium release assays. This level of sensitivity was observed because NCC and targets were sufficiently different in size such that they could be resolved by scatter plots. Using FCM, cytotoxicity was detected earlier and at lower E:T ratios than previously reported for chromium release assays. Although tilapia were reported previously to be not capable of lysing IM-9 targets by chromium release detection, the more sensitive method of FCM detected cytotoxicity using PI uptake. HL-60 lysis by tilapia NCC exhibited saturable kinetics but occurred at different times post-cocultivation.
Oumouna
M
M
Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, University of Georgia, Athens, Georgia 30602, USA.
Jaso-Friedmann
L
L
Evans
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DL
D000818:Animals; D017209:Apoptosis / Q000276:immunology; D002463:Cell Membrane Permeability / Q000276:immunology; D002469:Cell Separation; D002857:Chromium / Q000378:metabolism; D023681:Cichlids; D018920:Coculture Techniques; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D003602:Cytotoxicity, Immunologic; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D007694:Killer Cells, Natural / Q000166:cytology / Q000276:immunology* / Q000378:metabolism; D008027:Light; D008297:Male; D009336:Necrosis; D011419:Propidium / Q000378:metabolism; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity
D002857:Chromium; D011419:Propidium