Oner Ozdemir1. 1. Dept. of Pediatrics, İstanbul Göztepe Research and Training Hospital, Fahrettin Kerim Gökay Caddesi, Kadıköy,İstanbul, Turkey. ozdemir_oner@hotmail.com
Abstract
BACKGROUND: Chromium release assay is the standard method for the evaluation of cell-mediated cytotoxicity including mast cell's in vitro. Although this is a reproducible method, it has more drawbacks than radioactivity. In addition to the shortcoming of measuring just necrotic killing, some non-radioactive methods have also not been widely accepted and available. OBJECTIVE: This study describes a flow cytometric approach measuring mast cell-mediated cytotoxicity with marking target cells by monoclonal antibody, besides Annexin V/PI co-labeling to detect cytotoxicity. METHODS: A colony forming unit mast in vitro was developed from human bone marrow mononuclear cells in serum-free methylcellulose medium. 8-week-old mast cells as effectors and Daudi/Raji cells as targets were utilized. They both were co-incubated in certain ratios for short/long term. Percent/Cumulative cytotoxicity was calculated by this method. For verification and comparison, experiments were repeated and chromium release assay was done, respectively. RESULTS: This method clearly was able to show human mast cell-mediated cytotoxicity against human tumors. Moreover, this technique also allowed us to separate different stages of cytotoxic killing as early and late apoptotic. There were statistically significant correlations among percent and cumulative cytotoxicity, and chromium release assay. Standard NK-/LAK-sensitive cells were found to be very susceptible to mast cell cytotoxicity that happened in short term as well. CONCLUSIONS: This technique is reliable, is more sensitive and has some advantages no less than the evaluation of early apoptosis and exact total cell loss. Our findings suggest MC's anti-tumor role and these in vitro studies help enlighten MC's interactions with tumor cells.
BACKGROUND: Chromium release assay is the standard method for the evaluation of cell-mediated cytotoxicity including mast cell's in vitro. Although this is a reproducible method, it has more drawbacks than radioactivity. In addition to the shortcoming of measuring just necrotic killing, some non-radioactive methods have also not been widely accepted and available. OBJECTIVE: This study describes a flow cytometric approach measuring mast cell-mediated cytotoxicity with marking target cells by monoclonal antibody, besides Annexin V/PI co-labeling to detect cytotoxicity. METHODS: A colony forming unit mast in vitro was developed from human bone marrow mononuclear cells in serum-free methylcellulose medium. 8-week-old mast cells as effectors and Daudi/Raji cells as targets were utilized. They both were co-incubated in certain ratios for short/long term. Percent/Cumulative cytotoxicity was calculated by this method. For verification and comparison, experiments were repeated and chromium release assay was done, respectively. RESULTS: This method clearly was able to show human mast cell-mediated cytotoxicity against humantumors. Moreover, this technique also allowed us to separate different stages of cytotoxic killing as early and late apoptotic. There were statistically significant correlations among percent and cumulative cytotoxicity, and chromium release assay. Standard NK-/LAK-sensitive cells were found to be very susceptible to mast cell cytotoxicity that happened in short term as well. CONCLUSIONS: This technique is reliable, is more sensitive and has some advantages no less than the evaluation of early apoptosis and exact total cell loss. Our findings suggest MC's anti-tumor role and these in vitro studies help enlighten MC's interactions with tumor cells.