Salari H, Takei F, Miller R, Chan-Yeung M
J Immunol Methods. 1987 Jun 26;100(1-2):91-7. doi: 10.1016/0022-1759(87)90176-1.
A novel technique for isolation of human lung mast cells is developed. Human lung tissue was enzymatically digested and the cells were partially purified by centrifugation on Percoll density gradient. Cells obtained at the Percoll density of 1.05-1.09 g/ml were then subjected to a cell sorter equipped with a single argon laser beam (FACS 440). Using four criteria as density, granularity, size and autofluorescence, four major cell populations were identified. One of the major populations contained 70-95% mast cells with a mean and SE values of 88 +/- 11% purity, n = 18 as determined by the measurement of total histamine content, light microscopic observation of stained cells with toluidine blue and estrase, and surface-stained IgE fluorescence antibody. Approximately less than 10% mast cells were identified in the three other major cell populations. Mast cells isolated by FACS were found to be intact, viable (approximately equal to 90%) and functionally normal as determined by the release of histamine evoked after stimulation with ionophone A23187, or challenged with anti-human IgE.
开发了一种分离人肺肥大细胞的新技术。对人肺组织进行酶消化，然后通过在Percoll密度梯度上离心对细胞进行部分纯化。然后将在Percoll密度为1.05 - 1.09 g/ml时获得的细胞置于配备单氩激光束的细胞分选仪（FACS 440）中。利用密度、粒度、大小和自发荧光这四个标准，鉴定出四个主要细胞群体。其中一个主要群体含有70 - 95%的肥大细胞，通过测量总组胺含量、用甲苯胺蓝和酯酶对染色细胞进行光学显微镜观察以及表面染色的IgE荧光抗体测定，其纯度的均值和标准误为88±11%，n = 18。在其他三个主要细胞群体中鉴定出的肥大细胞约少于10%。通过流式细胞仪分选分离出的肥大细胞被发现是完整、有活力的（约90%），并且通过用离子霉素A23187刺激后组胺的释放或用抗人IgE攻击来确定其功能正常。
