Evaluation of human mast cell-mediated cytotoxicity by DIOC18 target cell labeling in flow cytometry.
(51)Cr release assay (CRA) is still the standard method to study mast cell (MC)-mediated cytotoxicity in vitro. Non-radioactive methods e.g. MTT, Hoechst 22147 staining, have also been used. Though CRA has the benefit of being reproducible, it has several drawbacks e.g. spontaneous release and radioactivity. The basic strategy of this new flow cytometric assay involves labeling target cells with DIOC18, in addition to staining with propidium iodide to identify dead cells. 8-week-old human MCs were used as effectors. Human LAK-sensitive K-562; and LAK-resistant myeloid leukemia cell lines (DAMI, HL-60 and Meg-01) as well as 6 LAK-resistant myeloid leukemia patient samples were utilized. MCs/targets were co-incubated in certain ratios for short/long-term. Although there was some insignificant killing at 2 h/18 h, probably due to small sample size, significant killing in Meg-01 and HL-60 cells (27% and 39%; respectively) was observed at 48 h. This method clearly showed human MC-mediated cytotoxicity against human tumor cells. It was reproducible, reliable and cheaper without any radiation and spontaneous release. This is the first study to elucidate MC-mediated cytotoxicity by a flow cytometric assay.
Ozdemir
Oner
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Division of Allergy and Immunology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, 5th floor, Location C, ML 2000, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA. Oner.Ozdemir@cchmc.org
D002232:Carbocyanines; D002469:Cell Separation; D002478:Cells, Cultured; D003601:Cytotoxicity Tests, Immunologic; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D020014:K562 Cells; D008407:Mast Cells / Q000276:immunology* / Q000378:metabolism*
D002232:Carbocyanines; C098044:3,3'-dioctadecyloxacarbocyanine