微小染色体（<3 Mb）的流式分析与分选
Flow analysis and sorting of microchromosomes (<3 Mb).
作者信息
Ng Bee L, Yang Fengtang, Carter Nigel P
机构信息
The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, United Kingdom.
出版信息
Cytometry A. 2007 Jun;71(6):410-3. doi: 10.1002/cyto.a.20394.
BACKGROUND
The analysis and isolation of high numbers of chromosomes smaller than 3 Mb in size (microchromosomes) with good purity is dependent primarily on the detection sensitivity of the flow cytometer and the precision of the sort unit. The aim of this study was to investigate the capability of using a conventional flow cytometer for the detection and sorting at high purity microchromosomes with an estimated size of 2.7 Mb.
METHODS
Chromosomes were isolated from a human cell line containing a pair of X-derived microchromosomes, using a modified polyamine isolation buffer. The chromosome preparation was labeled with Hoechst and Chromomycin and analyzed and purified using a MoFlo sorter (DAKO) configured for high-speed sorting. The purity of the flow-sorted microchromosomes was assessed by reverse chromosome painting.
RESULTS
Improved resolution of the peak of microchromosomes in a bivariate plot of Hoechst versus Chromomycin fluorescence was obtainable after discriminating clumps and debris based on gating data within a FSC versus pulse width plot.
CONCLUSIONS
Chromosomes of smaller size, less than 3 Mb, can be detected with high resolution and flow-sorted with high purity using a conventional flow sorter.
背景
要分析和分离大量大小小于3 Mb的染色体（微染色体）并保证高纯度，主要取决于流式细胞仪的检测灵敏度和分选单元的精度。本研究的目的是探讨使用传统流式细胞仪检测和分选估计大小为2.7 Mb的高纯度微染色体的能力。
方法
使用改良的多胺分离缓冲液从含有一对X衍生微染色体的人类细胞系中分离染色体。染色体标本用Hoechst和色霉素标记，并用配置用于高速分选的MoFlo分选仪（达科公司）进行分析和纯化。通过反向染色体涂染评估流式分选微染色体的纯度。
结果
基于FSC与脉冲宽度图中的门控数据区分团块和碎片后，在Hoechst与色霉素荧光的双变量图中可获得微染色体峰的分辨率提高。
结论
使用传统的流式分选仪可以高分辨率检测小于3 Mb的较小染色体，并以高纯度进行流式分选。
Zotero 插件