Ozdemir Oner
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.
J Immunol Methods. 2007 Jan 30;319(1-2):98-103. doi: 10.1016/j.jim.2006.11.004. Epub 2006 Dec 8.
(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.
（51）铬释放试验（CRA）仍然是体外研究肥大细胞（MC）介导的细胞毒性的标准方法。也使用了非放射性方法，例如MTT、Hoechst 22147染色。虽然CRA具有可重复性的优点，但它有几个缺点，例如自发释放和放射性。这种新的流式细胞术检测的基本策略包括用二辛基碳菁碘（DIOC18）标记靶细胞，此外还用碘化丙啶染色以识别死细胞。使用8周龄的人肥大细胞作为效应细胞。使用对人淋巴因子激活的杀伤细胞（LAK）敏感的K-562；以及对LAK有抗性的髓系白血病细胞系（DAMI、HL-60和Meg-01）以及6份对LAK有抗性的髓系白血病患者样本。肥大细胞/靶细胞以一定比例短期/长期共孵育。虽然在2小时/18小时时有一些不显著的杀伤作用，可能是由于样本量小，但在48小时时观察到对Meg-01和HL-60细胞有显著杀伤作用（分别为27%和39%）。该方法清楚地显示了人肥大细胞对人肿瘤细胞的细胞毒性。它具有可重复性、可靠性且成本较低，无任何辐射和自发释放。这是第一项通过流式细胞术检测阐明肥大细胞介导的细胞毒性的研究。
