Cappella Paolo, Gasparri Fabio, Pulici Maurizio, Moll Jürgen
Department of Biology, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan, Italy.
Department of Chemistry, Drug Discovery Oncology, Nerviano Medical Sciences Srl, Milan, Italy.
Curr Protoc Cytom. 2015 Apr 1;72:7.34.1-7.34.17. doi: 10.1002/0471142956.cy0734s72.
Determination of incorporation of the thymidine analog 5-bromo-2'-deoxyuridine (BrdU) into DNA is a widely used method to analyze the cell cycle. However, DNA denaturation is required for BrdU detection with the consequence that most protein epitopes are destroyed and their immunocytochemical detection for multiplex analysis is not possible. A novel assay is presented for identifying cells in active S-phase that does not require the DNA denaturation step but nevertheless detects BrdU. For this purpose, cells were pulsed for a short time by 5-ethynyl-2'-deoxyuridine (EdU) which is incorporated into DNA. The nucleotide-exposed ethynyl residue was then derivatized by a copper-catalyzed cycloaddition reaction ("click chemistry" coupling) using a BrdU azide probe. The resulting DNA-bound bromouracil moieties were then detected by commercial anti-BrdU monoclonal antibodies without the need for a denaturation step. This method has been tested using several cell lines and is more sensitive than traditional BrdU and allows multicolor and multiplex analysis in flow cytometry (FCM) and image-based cytometry.
测定胸苷类似物5-溴-2'-脱氧尿苷（BrdU）掺入DNA的情况是一种广泛用于分析细胞周期的方法。然而，BrdU检测需要进行DNA变性，结果是大多数蛋白质表位被破坏，无法进行多重分析的免疫细胞化学检测。本文介绍了一种新的检测方法，用于识别处于活跃S期的细胞，该方法不需要DNA变性步骤，但仍能检测BrdU。为此，用5-乙炔基-2'-脱氧尿苷（EdU）对细胞进行短时间脉冲处理，EdU会掺入DNA。然后，使用BrdU叠氮化物探针通过铜催化的环加成反应（“点击化学”偶联）对核苷酸暴露的乙炔基进行衍生化。然后，无需变性步骤，通过商业抗BrdU单克隆抗体检测所得与DNA结合的溴尿嘧啶部分。该方法已在多种细胞系中进行了测试，比传统的BrdU更灵敏，并且允许在流式细胞术（FCM）和基于图像的细胞术中进行多色和多重分析。
