Flow cytometry and FISH to measure the average length of telomeres (flow FISH)
Flow cytometry and FISH to measure the average length of telomeres (flow FISH)
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DOI：
10.1038/nprot.2006.263
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发表时间：
2006-01-01
期刊：
影响因子：
14.8
通讯作者：
Lansdorp, Peter M.
中科院分区：
文献类型：
--
作者：
Baerlocher, Gabriela M.;Vulto, Irma;Lansdorp, Peter M.
端粒已经成为衰老和包括癌症在内的各种疾病的关键细胞元件。为了测量细胞中端粒重复序列的平均长度，我们描述了使用针对端粒重复序列的标记多肽核酸(PNA)探针的荧光原位杂交(FISH)结合流式细胞仪(Flow Cytotics)的荧光测量(Flow FISH)的方法。Flow FISH分析可以使用商业上可用的流式细胞仪进行，并且与其他测量端粒长度的方法相比具有独特的优势，因为它提供了关于数千个单个细胞中端粒重复长度的多参数信息。大多数移液(吸入和分配)步骤的自动化，以及在每个试管中包括具有已知端粒长度的内部标准(对照细胞)，提高了测量的准确性和重复性。分析22个不同个体的有核血细胞的基本方法需要大约12小时，分布在2-3天内。
Telomeres have emerged as crucial cellular elements in aging and various diseases including cancer. To measure the average length of telomere repeats in cells, we describe our protocols that use fluorescent in situ hybridization (FISH) with labeled peptide nucleic acid (PNA) probes specific for telomere repeats in combination with fluorescence measurements by flow cytometry (flow FISH). Flow FISH analysis can be performed using commercially available flow cytometers, and has the unique advantage over other methods for measuring telomere length of providing multi-parameter information on the length of telomere repeats in thousands of individual cells. The accuracy and reproducibility of the measurements is augmented by the automation of most pipetting (aspiration and dispensing) steps, and by including an internal standard (control cells) with a known telomere length in every tube. The basic protocol for the analysis of nucleated blood cells from 22 different individuals takes about 12 h spread over 2 - 3 days.