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細胞增殖檢測試劑盒中文說明（內容）：

1.1000X BrdU溶液：一個30µL小瓶10 mM BrdU。

2.抗BrdU單克隆抗體：一瓶10µL小鼠抗BrdU抗體。

3.固定/變性溶液：一瓶20 mL。

4.抗體稀釋劑：一瓶50 mL。

5.二抗體，HRP綴合物：一個20µL小瓶。

6.10X洗滌緩沖液：一瓶100 mL。

7.基質溶液：一個12 mL琥珀瓶。

8.停止溶液：一瓶12 mL。

艾美捷BrdU細胞增殖檢測試劑盒檢測原理：

該試劑盒檢測摻入細胞DNA的BrdU在使用抗BrdU抗體的細胞增殖期間。當細胞在含有BrdU，嘧啶類似物取代胸苷被并入新合成的DNA中增殖細胞（下圖）。一旦去除標記介質，細胞就被固定，DNA就被用固定/變性溶液一步變性（需要變性DNA以改善并入的BrdU用于檢測的可接近性）。然后是抗BrdU小鼠單克隆抗體加入抗體，然后加入HRP綴合的二抗體以檢測摻入的BrdU。顯影顏色的吸光度大小與BrdU的量成正比并可與細胞增殖直接相關。

圖：細胞選擇示意圖™ BrdU增殖ELISA

BrdU細胞增殖檢測試劑盒中的小鼠抗BrdU 單克隆抗體可以與摻入進DNA 的結合，再加入HRP 標記的羊抗小鼠，后通過TMB 底物液顯色，其顯色值高低與BrdU 摻入細胞量成正比。

相關文獻：

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.

來源：https://www.amyjet.com/products/CBA-251.shtml