Quantum dots thermal stability improves simultaneous phenotype-specific telomere length measurement by FISH-flow cytometry
Quantum dots thermal stability improves simultaneous phenotype-specific telomere length measurement by FISH-flow cytometry
复制标题
DOI：
10.1016/j.jim.2009.02.004
复制
发表时间：
2009-05-15
影响因子：
2.2
通讯作者：
Telford, William G.
中科院分区：
文献类型：
--
作者：
Kapoor, Veena;Hakim, Fran T.;Telford, William G.
端粒长度分析已大大简化了定量流式细胞术技术FISH-流。在该方法中，将与端粒末端重复序列互补的荧光素标记的合成寡核苷酸与端粒序列杂交，并通过流式细胞术测量所得荧光。这种技术已经取代了传统的费力的Southern印迹端粒长度测量技术在许多实验室，并允许高通量样品格式的端粒长度的单细胞分析。然而，端粒探针退火所需的苛刻条件（82 ℃）使得难以成功地将该技术与同时免疫标记联合收割机结合。大多数传统的有机荧光探针（即荧光素、藻红蛋白等）尽管努力共价交联抗原-抗体-荧光团复合物，但其热稳定性有限，并且无法经受高温退火过程。探针荧光的这种损失使得难以测量复杂淋巴细胞群体中的FISH流，并且通常迫使研究人员使用荧光激活的细胞分选来预分离它们的群体，这是一种需要大量细胞的费力技术。在这项研究中，我们用量子点（纳米颗粒）取代了FISH流中的传统荧光团。量子点被证明具有比传统的低分子量和藻胆蛋白荧光团更大的热稳定性。发现针对单核细胞和T细胞抗原的量子点抗体缀合物在高温退火步骤后保留其大部分荧光，从而允许同时进行荧光免疫表型分析和端粒长度测量。由于量子点具有非常窄的发射带宽，我们能够同时分析多个量子点抗体缀合物（Qdot 605、655和705）与FISH流测量，以评估人单核细胞和T细胞亚群中端粒长度的年龄相关下降。使用量子点免疫标记，计算出CD 4+细胞端粒长度的平均减少率为41.8 bp/年，非常接近先前使用传统流式FISH和Southern印迹法报道的值。因此，对传统流式FISH技术的这种修改应该允许同时进行荧光免疫表型分析和端粒长度测量，从而允许在少量细胞中进行复杂的细胞亚群特异性分析，而不需要预先进行细胞分选。由爱思唯尔公司出版
Telomere length analysis has been greatly simplified by the quantitative flow cytometry technique FISH-flow. In this method, a fluorescein-labeled synthetic oligonucleotide complementary to the telomere terminal repeat sequence is hybridized to the telomere sequence and the resulting fluorescence measured by flow cytometry. This technique has supplanted the traditional laborious Southern blot telomere length measurement techniques in many laboratories, and allows single cell analysis of telomere length in high-throughput sample formats. Nevertheless, the harsh conditions required for telomere probe annealing (82 degrees C) has made it difficult to successfully combine this technique with simultaneous immunolabeling. Most traditional organic fluorescent probes (i.e. fluorescein, phycoerythrin, etc.) have limited thermal stability and do not survive the high temperature annealing process, despite efforts to covalently crosslink the antigen-antibody-fluorophore complex. This loss of probe fluorescence has made it difficult to measure FISH-flow in complex lymphocyte populations, and has generally forced investigators to use fluorescent-activated cell sorting to pre-separate their populations, a laborious technique that requires prohibitively large numbers of cells. In this study, we have substituted quantum dots (nanoparticles) for traditional fluorophores in FISH-flow. Quantum dots were demonstrated to possess much greater thermal stability than traditional low molecular weight and phycobiliprotein fluorophores. Quantum dot antibody conjugates directed against monocyte and T cell antigens were found to retain most of their fluorescence following the high temperature annealing step, allowing simultaneous fluorescent immunophenotyping and telomere length measurement. Since quantum dots have very narrow emission bandwidths, we were able to analyze multiple quantum dot antibody conjugates (Qdot 605, 655 and 705) simultaneously with FISH-flow measurement to assess the age-associated decline in telomere length in both human monocytes and T cell subsets. With quantum dot immunolabeling, the mean decrease rate in telomere length for CD4+ cells was calculated at 41.8 bp/year, very close to previously reported values using traditional flow-FISH and Southern blotting. This modification to the traditional flow-FISH technique should therefore allow simultaneous fluorescent immunophenotyping and telomere length measurement, permitting complex cell subset-specific analysis in small numbers of cells without the requirement for prior cell sorting. Published by Elsevier B.V.