The investigation of cell cycle ａnd DNA synthesis has been essential to many fields of science.

Traditionally a radiolabled thymidine has been used to track new DNA synthesis ａnd cellular

proliferation. Although quite sensitive, use of radiolabled thymidine has the limitation of having to

regulate, handle ａnd dispose of radioisoes ａnd often requires expensive detection equipment.

More recently, the thymidine analog 5-bromo-2’-deoxyuridine (BrdU) has been used in place of

radiolabled thymidine ａnd is incorporated into newly synthesized DNA strands of actively proliferating cells. Following fixation ａnd partial denaturation of cellular DNA, BrdU can be detected

immunochemically which allows for the analysis of live cell new DNA synthesis.

Assay Principle

The CytoSelect™ BrdU Cell Proliferation ELISA Kit detects BrdU incorporated into cellular DNA

during cell proliferation using an anti-BrdU antibody. When cells are incubated in media containing

BrdU, the pyrimidine analog is incorporated in place of thymidine into the newly synthesized DNA of

proliferating cells (Figure 1). Once the labeling media is removed, the cells are fixed ａnd the DNA is

denatured in one step with a fix/denature solution (denaturation of the DNA is necessary to improve

the accessibility of the incorporated BrdU for detection). Then an anti-BrdU mouse monoclonal

antibody is added followed by an HRP conjugated secondary antibody to detect the incorporated BrdU.

The magnitude of the absorbance for the developed color is proportional to the quantity of BrdU

incorporated into cells ａnd can be directly correlated to cell proliferation.

艾美捷BrdU细胞增殖检测试剂盒中文说明（内容）：

1.1000X BrdU溶液：一个30µL小瓶10 mM BrdU。

2.抗BrdU单克隆抗体：一瓶10µL小鼠抗BrdU抗体。

3.固定/变性溶液：一瓶20 mL。

4.抗体稀释剂：一瓶50 mL。

5.二抗体，HRP缀合物：一个20µL小瓶。

6.10X洗涤缓冲液：一瓶100 mL。

7.基质溶液：一个12 mL琥珀瓶。

8.停止溶液：一瓶12 mL。

艾美捷BrdU细胞增殖检测试剂盒检测原理：

该试剂盒检测掺入细胞DNA的BrdU在使用抗BrdU抗体的细胞增殖期间。当细胞在含有BrdU，嘧啶类似物取代胸苷被并入新合成的DNA中增殖细胞（下图）。一旦去除标记介质，细胞就被固定，DNA就被用固定/变性溶液一步变性（需要变性DNA以改善并入的BrdU用于检测的可接近性）。然后是抗BrdU小鼠单克隆抗体加入抗体，然后加入HRP缀合的二抗体以检测掺入的BrdU。显影颜色的吸光度大小与BrdU的量成正比并可与细胞增殖直接相关。

图：细胞选择示意图™ BrdU增殖ELISA

BrdU细胞增殖检测试剂盒中的小鼠抗BrdU 单克隆抗体可以与掺入进DNA 的结合，再加入HRP 标记的羊抗小鼠，后通过TMB 底物液显色，其显色值高低与BrdU 掺入细胞量成正比。

相关文献：

1. Shoghi KI, Xu J, Su Y, He J, Rowland D, Yan Y, Garbow JR, Tu Z, Jones LA, Higashikubo R,

Wheeler KT, Lubet RA, Mach RH, You M.. (2013) PLoS One. 8:e74188

2. Gong F, Wang G, Ye J, Li T, Bai H, Wang W (2013) Oncol Rep. 30:2976-2982

3. Piastowska-Ciesielska AW, Kozłowski M, Wagner W, Domińska K, Ochędalski T. (2013) Arch

Med Sci. 30:739-744.

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