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

ABSTRACT

Background: Chromium 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. Methods: A 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. Results: This 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. Conclusions: When 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(AU)