Telomere length measurements in leukocyte subsets by automated multicolor flow-FISH
Telomere length measurements in leukocyte subsets by automated multicolor flow-FISH
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DOI：
10.1002/cyto.a.10064
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发表时间：
2003-09-01
期刊：
影响因子：
3.7
通讯作者：
Lansdorp, PM
中科院分区：
文献类型：
--
作者：
Baerlocher, GM;Lansdorp, PM
背景：端粒是染色体末端必需的蛋白质- dna结构，与基因组稳定性和细胞复制有关。端粒代表的平均长度可以通过原位杂交和流式细胞术来测量[flow- fish]。这样的端粒长度值反映了配子遗传的染色体特异性端粒长度的端粒缩短（由细胞分裂、氧化损伤等原因引起）和端粒延长（主要由端粒酶活性引起）。在这里，我们报告了flow-FISH方法的改进，该方法能够测量人有核血细胞亚群的端粒长度。方法和结果：为了测量血液样本中粒细胞、幼稚T细胞、记忆T细胞、B细胞和自然杀伤(NK)/NKT细胞的端粒长度，我们将flow-FISH与抗体染色（multi - color flow-FISH）相结合。染色方案中的大多数步骤使用96孔微分配器设备自动完成。端粒长度的最小可检测差异和该方法的重现性在0.2-0.5 kb的范围内，并且可以用少至1000个细胞进行测量。结论：自动化多色流动fish将极大地促进有核血细胞亚群端粒长度调节的研究，特别是当只有少量细胞可用和端粒长度差异很小的情况下。(C) 2003 Wiley-Liss, Inc。
Background: Telomeres are essential protein-DNA structures at the end of chromosomes which are implicated in genome stability and cell replication. The average length of telomere rep eats can be measured by in situ hybridization and flow cytometry [flow-FISH]. Such telomere length values reflect telomere shortening (resulting from cell divisions, oxidative damage and other causes) and telomere elongation (mainly resulting from telomerase activity) of the chromosome-specific telomere length inherited in the gametes. Here we report improvements in flow-FISH methodology that enable measurements of telomere length in subsets of human nucleated blood cells.Methods and Results: In order to measure the telomere length in granulocytes, naive T cells, memory T cells, B cells and natural killer (NK)/NKT cells within a blood sample, we combined flow-FISH with antibody-staining (Multicolor flow-FISH). Most steps in the staining protocol were automated using a 96-well microdispenser device. The minimum detectable difference in telomere length and the reproducibility of the method are in the range of 0.2-0.5 kb and measurements can be made with as few as a thousand cells.Conclusions: Automated multicolor flow-FISH will greatly facilitate studies of telomere length regulation in subsets of nucleated blood cells, especially when only few cells are available and when differences in telomere length are small. (C) 2003 Wiley-Liss, Inc.