Factors affecting flow karyotype resolution
Factors affecting flow karyotype resolution
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DOI：
10.1002/cyto.a.20330
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发表时间：
2006-09-01
期刊：
影响因子：
3.7
通讯作者：
Carter, Nigel P.
中科院分区：
文献类型：
--
作者：
Ng, Bee Ling;Carter, Nigel P.
背景：影响染色体纯度的主要因素之一，特别是在高速分选过程中，是单个染色体峰在流动核型中的分析分辨率，以及碎片和碎片染色体的数量。我们研究了染色体悬浮液制备过程中影响核型分辨的因素。方法：采用不同PH值、盐浓度和染色时间的多胺缓冲液分离方法，从不同类型的人和动物细胞中分离染色体。每种制剂在高速分选的MoFlo分选器（DAKO）上进行分析，并比较了流程核型的分辨率。结果：采用PH为8.0的低渗缓冲液和PH为7.50 ~ 8.0的多胺分离缓冲液（不含NaCl）分离染色体，获得了高分辨率的流式细胞仪数据。从经过足够中期停滞时间的细胞系中分离的染色体悬浮液，将染色时间延长至8小时以上，产生的碎片百分比最低，染色体峰更紧，总体变异系数更低，分离的染色体产量增加1至5倍。结论：缓冲液PH和染色长度的优化提高了核型分辨率，特别是对于较大的染色体，并减少了染色体片段（碎片）的存在。然而，最有趣和令人惊讶的发现是，PAB缓冲液中不含NaCl提高了较大染色体的产量和分辨率。
Background: One of the major factors which influences the chromosome purity achievable particularly during high speed sorting is the analytical resolution of individual chromosome peaks in the flow karyotype, as well as the amount of debris and fragmented chromosomes. We have investigated the factors involved in the preparation of chromosome suspensions that influence karyotype resolution.Methods: Chromosomes were isolated from various human and animal cell types using a series of polyamine buffer isolation protocols modified with respect to PH, salt concentration, and chromosome staining time. Each preparation was analyzed on a MoFlo sorter (DAKO) configured for high speed sorting and the resolution of the flow karyotypes compared.Results: High resolution flow cytometric data was obtained with chromosomes optimally isolated using hypotonic solution buffered at PH 8.0 and polyamine isolation buffer (with NaCl excluded) between PH 7.50 and 8.0. Extending staining time to more than 8 h with chromosome suspensions isolated from cell lines subjected to sufficient metaphase arrest times gave the best result with the lowest percentage of debris generated, tighter chromosome peaks with overall lower coefficients of variation, and a 1- to 5-fold increase in the yield of isolated chromosomes.Conclusions: Optimization of buffer PH and the length of staining improved karyotype resolution particularly for larger chromosomes and reduced the presence of chromosome fragments (debris). However, the most interesting and surprising finding was that the exclusion of NaCl in PAB buffer improved the yield and resolution of larger chromosomes.