Design of a flow cytometric assay for the determination of natural killer and cytotoxic T-lymphocyte activity in human and in different animal species.
BACKGROUND
The most common assay used to detect natural killer (NK) and cytotoxic T-lymphocyte (CTL) activity is the (51)Cr release assay. The numerous disadvantages of this method led us to evaluate cytotoxicity functions by flow cytometry. We described a flow cytometric assay to assess NK and CTL activity from different species.


METHODS
This assay is based on a dual fluorescent staining of target cells. The dye, DIOC18((3)) (3, 3'-dioctadecyloxacarbocyanine perchlorate), is used to stain the membrane of different target cells. Propidium iodide (PI) is used to label dead target and effector cells. This labeling allows a clear discrimination between both cell populations.


RESULTS
A good correlation was observed between the percentage of target lysis and the effector-to-target cell (E/T) ratios with human and porcine peripheral blood mononuclear cells (PBMC) as effector cells. The flow cytometric assay was shown to be as sensitive and as reliable as the (51)Cr release performed with human cells. The assay was also applied successfully to measure NK cell activity in other animal species (pig, rabbit, hen, and mouse) and to measure murine CTL activity against the influenza virus.


CONCLUSIONS
We provide evidence that the flow cytometric assay using DIOC18((3)) is highly reproducible and is suitable to measure different types of cell cytotoxicity.
Piriou
L
L
Agence Française de Sécurité Sanitaire des Aliments, Unité de Virologie et Immunologie Porcines, Ploufragan, France. l.piriou@ploufragan.afssa.fr
Chilmonczyk
S
S
Genetet
N
N
Albina
E
E
D000818:Animals; D016923:Cell Death; D002478:Cells, Cultured; D002645:Chickens; D002860:Chromium Radioisotopes; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D003602:Cytotoxicity, Immunologic / Q000276:immunology*; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes; D006801:Humans; D007114:Immunization; D007694:Killer Cells, Natural / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D009975:Orthomyxoviridae / Q000276:immunology; D011817:Rabbits; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013154:Spleen / Q000166:cytology / Q000276:immunology; D013552:Swine; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology*; D014407:Tumor Cells, Cultured; D014616:Vaccinia virus / Q000276:immunology
D002860:Chromium Radioisotopes; D005456:Fluorescent Dyes