TY - JOUR T1 - Measuring human mast cell-mediated cytotoxicity against human tumour cells: three-colour flow cytometry using monoclonal antibody target staining with annexin V / propidium iodide co-labelling of death JO - Allergologia et Immunopathologia T2 - AU - Özdemir,Ö. SN - 03010546 M3 - 10.1016/j.aller.2010.07.007 DO - 10.1016/j.aller.2010.07.007 UR - https://www.elsevier.es/en-revista-allergologia-et-immunopathologia-105-articulo-measuring-human-mast-cell-mediated-cytotoxicity-S0301054610002466 AB - BackgroundChromium release assay is the standard method for evaluation of cell-mediated cytotoxicity, including that of mast cells. Although this is a reproducible method, it has more drawbacks than even radioactivity. In addition to the shortcoming of measuring only necrotic killing, some non-radioactive methods have not been widely used either. The numerous limitations of these methods led researchers to develop other techniques. This study describes a new flow cytometric approach that measures human mast cell-mediated cytotoxicity by marking target cells with monoclonal antibody alongside annexin V/ PI co-labelling. MethodsA colony forming unit - mast in vitro was developed from human bone marrow mononuclear cells in serum-free methylcellulose medium. Six-week-old human bone marrow-derived mast cells were used as effectors, and malignant B-lymphoblastoid cell lines like Daudi / Raji cells as targets. Effectors and targets were both co-incubated for short and long-term durations, and experiments were repeated several times. Cytotoxicity was calculated by flow cytometric mast cell-mediated cytotoxicity assay. ResultsThis method was able to clearly show mast cell-mediated cytotoxicity against human tumours. It is well-known that some lymphokine-activated killer-sensitive cells are resistant to mast cell-mediated cytotoxicity. However, a different type of lymphokine activated killer-sensitive cell in this study was found to be very sensitive to mast cell-mediated cytotoxicity. Moreover, this technique also allowed us to separate killing into different stages: early and late apoptosis. ConclusionsWhen compared to chromium release and non-radioactive methods, this method has the advantages of allowing evaluation of early apoptosis and short/long term mast cell-mediated cytotoxicity with specific target marking. ER -