Cell Analysis – In Vivo EdU Cell Proliferation Assays

Cell analysis allows scientists to gain unique insights into cell proliferation, viability, and toxicity, enabling the monitoring of cell response and cell health in cultures after treatment with various stimuli.

Detection of DNA replication is a powerful tool for studying proliferation of both cell populations and individual cells. DNA replication rates can be detected through the incorporation of a nucleoside analog such as BrdU or EdU into DNA. The detection of these two thymidine analogs differs significantly and has implications for test results, potential applications, and multiplexing capability. Compared to other cell proliferation methods, the EdU proliferation assay does not involve radioactive isotopes or antibodies to detect newly synthesized DNA.

The ClickTech EdU cell proliferation assay enables estimates of cell proliferation rates by determining the fraction of cells replicating their DNA. The technology is an in vivo DNA labeling with EdU (5-ethynyl-2′-deoxyuridine), which is cell-permeable and can be administered by injection (intraperitoneal, intramuscular, subcutaneous), orally or by direct incubation in certain organisms (e.g. mice), wherein EdU is incorporated into actively dividing cells.

Schematic representation of EdU cell proliferation assays. A) Incubation of cells with EdU. B) EdU is incorporated during active DNA synthesis. C) Detection of cell proliferation via click chemistry, wherein the use of a range of different fluorescent dyes is possible.

Application

This assay enables monitoring genotoxicity, anticancer drug evaluation or cell cycle analysis by direct detection of DNA replication in proliferating cells. The principle of this procedure is similar to that of the BrdU assay, with the difference of the detection reaction. In summary, the thymidine analog, in this case the EdU (5-ethynyl-2′-deoxyuridine), is added to the cell culture media, taken up by the cells and incorporated into newly synthesized DNA during S-phase. This nucleoside contains a reactive alkyne group (instead of the bromine atom in BrdU) and can be conjugated to molecules bearing azide groups through a click reaction. Functional alkyne and azide groups do not occur in natural cell systems and do not react without catalytic reagents. This ensures that the EdU cell proliferation method, based on click chemistry, is highly selective, sensitive and reproducible.

After an incubation period that depends on the specific cell type, the cells are washed, fixed and permeabilized to allow the detection reagents to penetrate. The detection is then carried out via the copper-catalyzed click reaction (also known as Azide-Alkyne Cyclo-addition or CuAAC). Click reactions show high quantitative yields, run under mild conditions and are easy to perform. In order to start the reaction, a click cocktail, containing buffer, catalytic reagents and a fluorescent dye, is added to the cells. Only cells that actively replicate their DNA during the EdU pulse can be detected by this method.

This technology allows an almost unlimited choice of different and easy to synthesize fluorescent dyes and thus offers maximum flexibility in terms of “detection colors” and analytical devices. Cell proliferation can be detected by fluorescence microscopy, flow cytometry and fluorescence plate reader. baseclick currently offers assays for these three analytical methods and up to four standard dyes (488, 555, 594, 647 nm).

Product Overview

*The three kit sizes S, M and L with increasing EdU content can be selected depending on the animal to be examined or the number of animals.

LITERATURE

An efficient protocol for in vivo labeling of proliferating epithelial cells, C. Michel et al., 2018, J. Immunol. Methods, Vol. 457, p. 82-86.

https://doi.org/10.1016/j.jim.2018.03.015

Cisplatin-induced DNA double-strand breaks promote meiotic chromosome synapsis in PRDM9-controlled mouse hybrid sterility, L. Wang et al., 2018, eLife, Vol. 7, p. e42511.

https://doi.org/10.7554/eLife.42511

FXR Regulates Intestinal Cancer Stem Cell Proliferation, T. Fu et al., 2019, Cell, Vol. 176, p. 1098-1112.

https://doi.org/10.1016/j.cell.2019.01.036