EdU Cell Proliferation Assay for Imaging

Cell Analysis: Cell Proliferation Imaging Assays

The ClickTech EdU Cell Proliferation Kit for Imaging is a superior alternative to BrdU staining method, for measuring and quantifying de novo DNA synthesis during cell proliferation.

The EdU assay relies on the biorthogonal incorporation of thymidine analog 5-ethynyl-2`-desoxyuridine (5-EdU or EdU) into de novo synthesized DNA. As EdU is not endogenously present in biological molecules, cells, tissues, or model organisms, it can subsequently be conjugated with a bright, photostable dye using click chemistry technology, which was awarded with a Nobel prize in 2022. This reaction is fast, highly specific, and mild, enabling this cell proliferation detection in cancer biology, immunology, cell biology, and developmental biology.

ClickTech EdU Cell Proliferation Kit for Imaging contains chemicals and a protocol optimized for analysis based on fluorescent microscopy applications.

Challenges in traditional cell proliferation assays using 5-Bromodeoxyuridine (BrdU)

Traditional Cell proliferation assays are based on the incorporation of the thymidine analog 5-bromodeoxyuridine (5-BrdU or BrdU) into newly synthesized DNA. BrdU detection depends on anti-BrdU antibodies, which are unable to bind to BrdU within double stranded DNA. Therefore, DNA needs to be denatured to form single stranded DNA (ssDNA) before detection under harsh conditions such as HCl, heat or enzymes. The denaturation step is time consuming and difficult to perform consistently, additionally the necessary harsh treatment can affect sample integrity and quality.

EdU: An enhanced and efficient alternative for cell proliferation assays

The ClickTech EdU cell proliferation assays from baseclick provide an effective solution to the limitations and disadvantages of traditional methods. During the S-Phase of the cell cycle, 5-ethynyl-2′-deoxyuridine (EdU) is incorporated into DNA instead of thymidine during de novo synthesis. Due to the small size of the fluorescent azide, EdU is detected after DNA incorporation without the need for antibodies or DNA denaturation.

Moreover, the reagents provided in ClickTech EdU cell proliferation kits enable mild fixation and permeabilization of cells, allowing sufficient access to DNA for the small molecules used for EdU cell proliferation assays.

The EdU cell proliferation assay is optimized with an easy detection protocol for researchers, simplifying the workflow and the time required. The direct detection procedure for imaging applications is completed within 30 minutes and is compatible with multiplexing for content- and context-rich results.

Reliable and high-resolution cell proliferation analysis with ClickTech EdU Imaging Kit

The ClickTech EdU Imaging Kit provides a fast, consistent, and reliable method to evaluate cell proliferation across various research areas. It supports quantitative, high-resolution detection of replicating cells and is well-suited for imaging-based cell cycle analysis.

Fluorescence imaging of replicating HeLa cells detected with the EdU Cell Proliferation Kit for Imaging (BCK-EdU488IM100). Green signal: EdU labelling; blue signal: DNA counterstaining with DAPI; scale bar = 10 µm. Distinct replication patterns allow staging of replicating cells along S phase (early/mid/late S phase).

Cell proliferation assay with the EdU Cell Proliferation Kit for Imaging (BCK-EdU647IM100). A) Representative fluorescence microscopy image. Red signal: EdU labelling; blue signal: DNA counterstaining with DAPI. B) Cells in S, G1 and G2/M phases are quantified by image cytometry.

Applications:

• High-resolution analysis of cell proliferation in adherent cultures
• Assessment of drug-induced cell cycle effects in vitro
• Multiplex imaging with nuclear, cytoplasmic, or membrane markers
• Quantitative analysis of S-phase entry in fixed samples
• Research in cancer biology, toxicology, developmental biology, and regenerative medicine

Innovative features of the ClickTech EdU cell proliferation assay for enhanced DNA Synthesis detection

• Enables direct detection of DNA synthesis using EdU incorporation and click chemistry
• Antibody-free protocol ensures low background and preserves cell morphology
• Available in multiple fluorescent dyes for flexible experimental design
• Compatible with adherent cells and multiplex immunostaining workflows
• Simple and fast protocol – complete detection in under 30 minutes

LITERATURE

Anticancer drug and ionizing radiation-induced DNA damage differently influences transcription activity and DDR-related stress responses of an endothelial monolayer, V. Ziegler et al., 2020, Molecular Cell Research, Vol. 1867, p. 118678.

https://doi.org/10.1016/j.bbamcr.2020.118678

Dendrimer-Based Signal Amplification of Click-Labelled DNA in Situ, N. Raddaoui et al. 2017, ChemBioChem, Vol. 18, p. 1716-1720.

https://doi.org/10.1002/cbic.201700209

High-copy sequences reveal distinct evolution of the rye B chromosome, S. Klemme et al., 2013, New Phytologist, Vol. 199, p. 550-558.

https://doi.org/10.1111/nph.12289

Cytolethal Distending Toxin Promotes Replicative Stress Leading to Genetic Instability Transmitted to Daughter Cells, W. Tremblay et al., 2021, Frontiers in Cell and Developmental Biology, Vol. 9, 656795.

https://doi.org/10.3389/fcell.2021.656795

Brassica napus Roots Use Different Strategies to Respond to Warm Temperatures, M. Boter et al., 2023, Int. J. Mol. Sci., Vol. 24(2), 1143.

https://doi.org/10.3390/ijms24021143

BMP signaling promotes zebrafish heart regeneration via alleviation of replication stress., M. D. Vasudevarao et al., 2025, Nat Commun, Vol. 16, 1708.

https://doi.org/10.1038/s41467-025-56993-6