EdU Cell Proliferation Assays For Imaging, Flow Cytometry & High Throughput Screening
baseclick’s EdU Cell Proliferation Assays combine EdU (5-ethynyl 2´-deoxyuridine) labeling with click chemistry to provide an alternative to traditional BrdU staining methods for detecting and quantitating newly synthesized DNA. These assays measure cell proliferation by detecting the incorporation of the alkyne-modified nucleoside EdU into DNA using copper-catalyzed azide-alkyne click chemistry to attach fluorescent probes. These assays simplify the experimental process while providing flexibility to use a variety of fluorescent conjugates.
The Detection of Cell Proliferation is of Utmost Importance, e.g., for
- Assessing cell health
- Determining genotoxicity
- Evaluation anticancer drugs
Monitoring active DNA-synthesis in cells is the best indicator of proliferation. In the traditional BrdU assay, BrdU is incorporated into the newly synthesized DNA of proliferating cells which are identified using an anti-BrdU antibody. DNA denaturation using HCl, heat, or digestion with DNase is required to expose the incorporated BrdU to the anti-BrdU antibody for detection. This process can lead to unwanted artifacts due to the harsh conditions. Such severe treatments can result in inconsistent staining and decreased signal.
Detection of Cell Proliferation, e.g., with BrdU
- Harsh conditions required
- Denaturation of DNA
- Time consuming
Unlike BrdU, EdU can be easily detected after DNA incorporation due to the small size of the fluorescent azide and does not require an antibody or DNA denaturation for detection. Thus, use of baseclick’s EdU Cell Proliferation Assays preserve 3-D protein structure and function with reduced target interference better than traditional BrdU staining. With their mild reaction conditions, baseclick’s EdU Cell Proliferation Assays allow for more consistent results, faster protocols, and better signal to noise compared to an antibody-based BrdU cell proliferation assay.
Benefits of baseclick’s EdU Cell Proliferation Assays
- Fast & direct detection without an antibody
- Save researchers valuable time with assay times of about 1 ½ hours
- Mild conditions - No denaturation required
- Highly sensitive: preserving cell morphology, DNA integrity, antigen binding sites, and fluorescent signal for better DNA staining
- Easy performance
- Reproducible - Consistent staining from robust protocols
- Four fluorescent read-outs
baseclick has developed different EdU kits for microscopy imaging, flow cytometry, and high throughput screening. Each kit comes with an optimized experimental protocol and the required components for each application. EdU incorporation can be visualized using the microscopy imaging kits. The flow cytometry kits allow researchers to obtain quantitative detection of the labeled cells. The high throughput kits have been optimized for measurements using a plate reader. Also, individual cells can be studied using an automated microscope or high content imaging system for reviewing cell morphology.
baseclick’s EdU Cell Proliferation Assays - Only Five Easy Steps…
- Feeding and proliferation
- Cell fixation
- Click cocktail
- Analysis: Imaging, Flow Cytometry or High Throughput Screening
Furthermore, in vivo kits have been developed that include larger amounts of EdU. So far, EdU has been incorporated into the following model organisms: mouse, rat, C. elegans, cricket, chicken, zebra fish larva, and Arabidopsis.
baseclick kits utilize fluorescent probes to detect the incorporated EdU. Researchers can choose from four fluorescent probes: 6-FAM (ex: 496 nm, em: 516 nm), 5-TAMRA (ex: 546 nm, em: 579 nm), 5/6-Sulforhodamine 101 (ex: 584 nm, em: 603 nm) and Eterneon-Red 645 (ex: 643 nm, em: 662 nm). These probes can be multiplexed with other fluorescent probes including using antibodies for immunostaining, expanding experimental options.