title: A flow-cytometry based cytotoxicity assay using stained effector cells in combination with native target cells.
abstract: Flow-cytometry based assays for cellular cytotoxicity have established themselves widely over the last years. Discrimination of target and effector cells is critical for such assays. If scatter properties are not informative, the standard approach until now has been to label the target cells with a suitable fluorescent dye. However, this cannot be applied to a number of experimental settings, e.g. if one effector cell type is tested against several target cells, or if target cells do not incorporate the dye properly. Therefore, our goal was to develop a protocol based on the labelling of effector cells. For this purpose, we came around to using a membrane dye, DIOC18, which is not commonly used for flow-cytometric applications. This dye showed very stable membrane integration properties that allowed long-term coincubation periods (24 h) without leakage to neighbouring cells. The vitality and cytotoxic activity of the effector cells were not altered by staining. For the detection of dead cells, the intercalating DNA-dye 7-AAD was used. The spectral emission wavelengths of this combination also enable the additional use of PE-conjugated antibodies to surface antigens in three-color cytometry devices. Cytotoxicity values obtained by our protocol were highly correlated with values obtained by the chromium release assay at different E/T ratios and using several target cell lines. All in all, we present here an easy to handle protocol, which enables the precise determination of cellular cytotoxicity in various experimental settings.
authors.affiliation: Institute of Immunology and Transfusion Medicine, University of Lübeck School of Medicine, Ratzeburger Allee 160, Germany.
authors.forename: Maike
authors.initials: M
authors.lastname: Höppner
authors.forename: Jürgen
authors.initials: J
authors.lastname: Luhm
authors.forename: Peter
authors.initials: P
authors.lastname: Schlenke
authors.forename: Petra
authors.initials: P
authors.lastname: Koritke
authors.forename: Christoph
authors.initials: C
authors.lastname: Frohn
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D002232:Carbocyanines; D002462:Cell Membrane / Q000276:immunology / Q000378:metabolism; D002860:Chromium Radioisotopes; D004396:Coloring Agents; D003601:Cytotoxicity Tests, Immunologic / Q000379:methods*; D003609:Dactinomycin / Q000031:analogs & derivatives*; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D006801:Humans; D066298:In Vitro Techniques; D020014:K562 Cells; D007694:Killer Cells, Natural / Q000276:immunology / Q000378:metabolism
chemical_list: D018952:Antigens, CD34; D002232:Carbocyanines; D002860:Chromium Radioisotopes; D004396:Coloring Agents; D005456:Fluorescent Dyes; D003609:Dactinomycin; C098044:3,3'-dioctadecyloxacarbocyanine; C025942:7-aminoactinomycin D