title: 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.
authors.affiliation: Department of Pediatrics (3(rd) Clinic), Allergy-Immunology Division, İstanbul Göztepe Research and Training Hospital, Göztepe, Kadıköy, Türkiye. ozdemir oner@hotmail.com
authors.forename: O
authors.initials: O
authors.lastname: Ozdemir
mesh_terms: D017304:Annexin A5 / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000378:metabolism; D017209:Apoptosis; D001854:Bone Marrow Cells / Q000166:cytology; D045744:Cell Line, Tumor; D002469:Cell Separation; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic; D005240:Feasibility Studies; D005434:Flow Cytometry / Q000379:methods / Q000639:trends; D006801:Humans; D008407:Mast Cells / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000276:immunology / Q000378:metabolism* / Q000473:pathology; D011419:Propidium / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D017304:Annexin A5; D000911:Antibodies, Monoclonal; D011419:Propidium