EdU Staining – Modern Method for Cell Proliferation Analysis

5-Ethynyl-2`-deoxyuridine (EdU or 5-EdU) is a modified nucleoside analog to thymidine. EdU has been developed as a superior alternative for 5`-Bromo-2´-deoxyuridine (BrdU) for the detection of de novo DNA synthesis. Therefore, EdU staining kits have been developed for proof of cell proliferation. The EdU staining kits work by incorporating EdU into the newly synthesized DNA. EdU is then detected by labeling it with an azido-labeled fluorescent dye using the copper catalyzed azide alkyne cycloaddition (CuAAC) click chemistry (learn more about click chemistry). The main advantages of EdU staining in comparison to BrdU are a simplified procedure, higher sensitivity, a reduced background and versatility.

• Simplified procedure: EdU staining kits do not need, unlike BrdU kits, harsh conditions to denature DNA to expose BrdU for antibody detection. The click chemistry reaction used to detect EdU does not need denatured DNA and, therefore, it is much simpler and faster.
• Higher sensitivity: Through the biorthogonal reaction of azides and alkynes, which are not endogenously present in any biomolecule, to form triazoles the CuAAC detection reaction of newly synthesized DNA is highly specific, allowing mild conditions and consistent labeling. This results in high sensitivity and more reproducible yields.
• Reduced background: Trough the highly efficient click chemistry used in covalently labeling the DNA in combination with the easy wash away of excess labeling reagent, EdU staining leads to a strongly reduced background signal enhancing the accuracy of the experimental data.
• Versatility: EdU staining is compatible with a lot of different cell analysis protocols as antibody staining or other fluorescent dyes as cell nucleus staining dyes. Therefore, EdU cell proliferation kits are suitable for multiparameter analyses for different detection methods such as imaging, flow cytometry and high throughput analyses. baseclick is offering EdU staining kits for all this detection methods with different detection wavelengths.

Understanding the EdU Staining Mechanism

The thymidine analog EdU is incorporated into newly synthesized DNA instead of thymidine by DNA polymerases in the newly formed strand. Already a small concentration of EdU of just 0.1 µM can be sufficient for EdU incorporation. The EdU is afterwards detected by EdU staining. Therefore, the alkyne moiety of EdU reacts with an azide-containing fluorescent dye in a Cu(I) catalyzed click chemistry reaction. This reaction is considered biorthogonal and leads to a highly specific and efficient labeling of DNA under mild conditions.

Kit EdU Staining: Streamlined Commercial Solutions

Main components of the Kit EdU Staining

baseclick`s EdU staining kits contain all components necessary to perform the EdU cell proliferation assay optimized on analysis method. As analysis methods, imaging, flow cytometry and high-throughput screening. There are also EdU cell staining assays optimized on the cell proliferation detection of a specific cell as T-Cells available.

All these kits contain EdU, a solution of a fluorescent azido dye, the copper catalyst, buffer additive to activate the catalyst and solutions to fix and permeabilize cells. baseclick also provides these kits with extra amounts of EdU if whole organism should be labeled as in vivo kits.

Benefits of Kit EdU Staining Products

baseclick´s EdU staining kits are designed to provide standardized reagents to guarantee reproducibility of experiments. baseclick establishes the reproducibility of their EdU staining kits by producing, exemplary, solutions in great amounts for the EdU staining kits. This minimizes on the one hand systematic errors as scaling mistakes and on the other provides components which are aliquoted from the bulk for a high amount of EdU staining kits.

For Kits using EdU staining straightforward protocols has been developed in combination with ready to use components to guarantee minimized preparation time, an easy usage to enable nonspecialized people to handle EdU staining kits and consistency of experiments.

Selecting the Right Kit EdU Staining Product

There are different methods to analyze EdU based cell proliferation. The main analyzation techniques for EdU staining kits are based on microscopy or on flow cytometry (FC).

EdU staining kits for FC can analyze large cell populations quickly with high throughput and quantitative analyses. Depending on the available fluorescent laser in the flow cytometer and planned multiparameter analyses, different dyes with different absorption wavelengths can be chosen. The most common wavelength used are 488 nm, 555 nm, 594 nm and 647 nm.

EdU staining kits for microscopy analysis can be divided into kits for high throughput analysis, which are designed to perform large-scale experiments in formats as 96 well plates for drug screening and in EdU kits to visualize cell proliferation on single-cell level. Both these variants of EdU staining kits for analysis are available with dyes of different absorption wavelengths and can be used for multiplexing.

Advanced Applications of Kit EdU Staining

Kit EdU Staining Cytometry for Flow-Based Analysis

For EdU staining detection by flow cytometry, a single-cell suspension of the cells to be analyzed must be prepared. The cell concentration should be known and set to a concentration that allows a flow of 10⁵ to 10⁷ cells/mL to allow ideal working conditions of most flow cytometry machines. If there is a plan to perform multiplexing using EdU staining kits for flow cytometry, it is important to use the correct fluorophore for EdU labeling which is not interfering with absorbance or emitting areas of other colorants used during the experiment. When antibody based staining reactions should be performed, they need to be included in the protocol between the EdU feeding and the EdU labeling reaction. This is the case because the Cu(I) needed to catalyze the EdU staining CuAAC reaction is destroying the tertiary structures of the antibodies.

Kit EdU Staining Imaging for Visual Analysis

EdU staining kits for Imaging are compatible with different other cell analyses methods for multiplexing such as immunofluorescence or indirect antibodies. When antibody based staining reactions should be performed, they need to be included in the protocol between the EdU feeding and the EdU labeling reaction. This is the case because the Cu(I) needed to catalyze the EdU staining CuAAC reaction is destroying the tertiary structures of the antibodies. Quantum dots experiments should be performed after EdU staining detection.

Kit EdU Staining for High Throughput Screening Applications

EdU staining kits for high throughput analysis are designed for reactions in 96 well plates and provide the solutions to perform this EdU detection reactions. But since a high number of reactions need to be performed and reaction mixtures for the EdU staining reactions need to be prepared, there is a lot of pipetting work to be performed. Therefore, EdU staining kits for high throughput screening have a high potential for automatic pipetting to save time as well as to reduce the human error rate. If no automatic pipetting is available, the usage of multi-channel pipettes is a great help.

EdU staining kits for high throughput screening are of great value to test a lot of compounds simultaneously for their influence on cell proliferation for certain cells like cancer cells to detect e.g. anti-proliferative compounds for drug development.

Future Perspectives of EdU Staining

EdU staining assays have been optimized in several ways over the last years. Protocols have been developed to use EdU in vivo application to study tumor growth in live animal models or to study cell regeneration in tissues such as the liver, the skin or in the heart. EdU staining kits for high throughput kits have become an important factor in testing the effect of potential drugs on cell proliferation or cytotoxicity. It was also achieved to use EdU staining kits to detect neurogenesis in large brain insects such as Heliconiini butterflies or cockroaches. Additionally, the analyzation tools for EdU staining have been improved to allow enhanced visualization of EdU-labels and the range of potential applications has been expanded to e.g. formalin fixed, paraffin-embedded (FFPE) tissues.