激光流式细胞术和细胞分选分离的肺细胞富集群体的电镜鉴定和形态学保存:一项新技术。

D P Penney, J F Leary, R A Cooper, A Paxhia
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引用次数: 3

摘要

越来越需要验证通过激光流式细胞术和荧光激活细胞分选(FACS)富集的细胞亚群的身份。当从整个器官或组织中分离的细胞亚群具有相似的特征(例如,大小,粒度,染色)时,光,相对比或荧光显微镜可能无法提供足够的分辨率来准确识别分离的细胞,并且许多流式细胞术参数(例如,活力,荧光)要求细胞在细胞横切激光束的分析点上是活的。在一些研究中,通过荧光显微镜鉴定为高度富集亚群的细胞通过电子显微镜发现含有其他细胞类型的显著群体。一种技术,固定在流动(FIF),已经发展起来,以增加能力相关联的形态学和激光分析细胞亚群。用固定液代替鞘液,允许在激光束分析活细胞后立即开始固定。这种新方法提高了恢复细胞亚群的细胞结构保存,以便通过透射或扫描电子显微镜进行评估。本报告介绍了应用该方法更准确地识别远端肺细胞亚群的结果,特别是II型肺细胞、Clara细胞和肺巨噬细胞。该方法的修改被用于从体外培养的小鼠肺成纤维细胞中分离成纤维细胞亚群,并且该方法被用于确定培养成纤维细胞对其他排列(例如x射线照射,细胞因子)的反应。
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Electron microscopic identification and morphologic preservation of enriched populations of lung cells isolated by laser flow cytometry and cell sorting: a new technique.

There is increasing need to verify the identities of cell subpopulations enriched by laser flow cytometry and fluorescence-activated cell sorting (FACS). When cell subpopulations isolated from whole organs or tissues have similar characteristics (e.g., size, granularity, staining), light, phase contrast or fluorescence microscopy may not provide sufficient resolution to identify isolated cells accurately and many flow cytometric parameters (e.g., viability, fluorescence) require the cells to be live at the point of analysis where the cell transects the laser beam. In some studies, cells identified by fluorescence microscopy as a highly enriched subpopulation were found by electron microscopy to contain significant populations of other cell types. A technique, fixation-in-flow (FIF), has been developed to increase ability to correlate morphological and laser analyses of cell subpopulations. Sheath fluid is replaced by fixative, permitting fixation to be initiated immediately after laser beam analysis of live cells. This new procedure yields improved cytoarchitectural preservation of recovered cell subpopulation(s) for evaluation by transmission or scanning electron microscopy. This report presents results from applying the methodology to identify more accurately cell subpopulations of the distal lung, specifically type II pneumocytes, Clara cells and pulmonary macrophages. A modification of this procedure was employed to isolate fibroblast subpopulations from murine lung fibroblasts grown in vitro and the procedure is being used to determine the responses of cultured fibroblasts to other permutations (e.g., X-irradiation, cytokines).

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