The Invitrogen Click-iT 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. Click-iT EdU assays simplify the experimental process while providing flexibility to use a variety of fluorescent conjugates. Click chemistry is important in numerous applications, including flow cytometry, immunohistochemistry, high content screening and fluorescent imaging techniques.

Benefits include:

  • Fast—Detection in as little as 80 min, without an antibody
  • Mild conditions—No denaturation required
  • Reproducible—Consistent staining from robust protocols

View cell proliferation assay protocols

Click-iT EdU selection guide

Click-iT EdU assays for flow cytometry

 Click-iT Plus EdU Alexa Fluor 350 Flow Cytometry KitClick-iT Plus EdU Pacific Blue Flow Cytometry Kit Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry KitClick-iT Plus EdU Alexa Fluor 594 Flow Cytometry KitClick-iT Plus EdU Alexa Fluor 647 Flow Cytometry Kit
Fluorescent labelAlexa Fluor 350 picolyl azidePacific Blue picolyl azideAlexa Fluor 488 picolyl azideAlexa Fluor 594 picolyl azideAlexa Fluor 647 picolyl azide
Laser (nm)UV405488532 or 561633/635
Ex/Em (nm)350/438410/455495/519532 or 561/615650/668
Sample typeOptimized for live cells, the click detection step comes after the fixation.
Bibliography
MultiplexableCan be co-stained with standard organic dyes (FITC, Alexa Fluor dyes, etc.); R-PE, R-PE tandems (such as R-PE-Cy5 or R-PE-Cy7); Fluorescent proteins (such as GFP and mCherry)
ReadoutHistogram may show separation of cells in S phase (DNA synthesis, including EdU incorporation)
Format50 assays50 assays50 assays100 assays50 assays50 assays100 assays
Cat. No. C10645C10636C10632C10633C10646C10634C10635
 Click-iT EdU Pacific Blue Flow Cytometry Kit Click-iT EdU Alexa Fluor 488 Flow Cytometry KitClick-iT EdU Alexa Fluor 647 Flow Cytometry Kit
Fluorescent labelPacific Blue azideAlexa Fluor 488 azideAlexa Fluor 647 azide
Laser405 nm488 nm633/635 nm
Ex/Em (nm)410/455495/519650/668
Sample typeOptimized for live cells, the click detection step comes after the fixation.
Bibliography
MultiplexableLimited multiplexability; can be co-stained with standard organic dyes (FITC, Alexa Fluor dyes, etc.). Not compatible with R-PE, R-PE tandems (such as R-PE-Cy5 or R-PE-Cy7); fluorescent proteins
ReadoutHistogram may show separation of cells in S phase (DNA synthesis, including EdU incorporation)
Format50 assays50 assays100 assays50 assays100 assays
Cat. No. C10418C10424C10420C10425C10419
 Click-iT Plus EdU Alexa Fluor 350 Flow Cytometry KitClick-iT Plus EdU Pacific Blue Flow Cytometry Kit Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry KitClick-iT Plus EdU Alexa Fluor 594 Flow Cytometry KitClick-iT Plus EdU Alexa Fluor 647 Flow Cytometry Kit
Fluorescent labelAlexa Fluor 350 picolyl azidePacific Blue picolyl azideAlexa Fluor 488 picolyl azideAlexa Fluor 594 picolyl azideAlexa Fluor 647 picolyl azide
Laser (nm)UV405488532 or 561633/635
Ex/Em (nm)350/438410/455495/519532 or 561/615650/668
Sample typeOptimized for live cells, the click detection step comes after the fixation.
Bibliography
MultiplexableCan be co-stained with standard organic dyes (FITC, Alexa Fluor dyes, etc.); R-PE, R-PE tandems (such as R-PE-Cy5 or R-PE-Cy7); Fluorescent proteins (such as GFP and mCherry)
ReadoutHistogram may show separation of cells in S phase (DNA synthesis, including EdU incorporation)
Format50 assays50 assays50 assays100 assays50 assays50 assays100 assays
Cat. No. C10645C10636C10632C10633C10646C10634C10635
 Click-iT EdU Pacific Blue Flow Cytometry Kit Click-iT EdU Alexa Fluor 488 Flow Cytometry KitClick-iT EdU Alexa Fluor 647 Flow Cytometry Kit
Fluorescent labelPacific Blue azideAlexa Fluor 488 azideAlexa Fluor 647 azide
Laser405 nm488 nm633/635 nm
Ex/Em (nm)410/455495/519650/668
Sample typeOptimized for live cells, the click detection step comes after the fixation.
Bibliography
MultiplexableLimited multiplexability; can be co-stained with standard organic dyes (FITC, Alexa Fluor dyes, etc.). Not compatible with R-PE, R-PE tandems (such as R-PE-Cy5 or R-PE-Cy7); fluorescent proteins
ReadoutHistogram may show separation of cells in S phase (DNA synthesis, including EdU incorporation)
Format50 assays50 assays100 assays50 assays100 assays
Cat. No. C10418C10424C10420C10425C10419

Click-iT EdU assays for imaging applications and microscopy

 Click-iT Plus EdU Alexa Fluor 488 Imaging KitClick-iT Plus EdU Alexa Fluor 555 Imaging KitClick-iT Plus EdU Alexa Fluor 594 Imaging KitClick-iT Plus EdU Alexa Fluor 647 Imaging Kit
Fluorescent labelAlexa Fluor 488 picolyl azideAlexa Fluor 555 picolyl azideAlexa Fluor 594 picolyl azideAlexa Fluor 647 picolyl azide
Standard filter setFITCTRITCTexas RedCy5
Ex/Em (nm)495/519555/565590/617650
Signal-to-noise ratio
Photostability
BibliographyCitations
ProtocolMicroscopy
Sample typeOptimized for live cells and tissue sections; the click detection step comes after the fixation
MultiplexableCompatible with antibody-fluorophore labeling including standard organic dyes (FITC, Alexa Fluor dyes) and fluorescent proteins such as GFP and mCherry. Do not use with phalloidin.
ReadoutFluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period
Format1 kit/50–250 coverslips1 kit/50–250 coverslips1 kit/50–250 coverslips1 kit/50–250 coverslips
Cat. No.C10637C10638C10639C10640
 Click-iT EdU Alexa Fluor 488 Imaging Kit Click-iT EdU Alexa Fluor 555 Imaging KitClick-iT EdU Alexa Fluor 594 Imaging KitClick-iT EdU Alexa Fluor 647 Imaging Kit
Fluorescent labelAlexa Fluor 488 azideAlexa Fluor 555 axideAlexa Fluor 594 azideAlexa Fluor 647 azide
Standard filter setFITCTRITCTexas RedCy5
Ex/Em (nm)495/519555/565590/617650/668
Signal-to-noise ratio
Photostability
BibliographyCitations
ProtocolMicroscopy
Sample typeOptimized for live cells and tissue sections; the click detection step comes after the fixation
MultiplexableCompatible with antibody-fluorophore labeling including standard organic dyes (FITC, Alexa Fluor dyes). Do not use with fluorescent proteins or phalloidin. 
ReadoutFluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period
Format1 kit/50–250 coverslips1 kit/50–250 coverslips1 kit/50–250 coverslips1 kit/50–250 coverslips
Cat. No.C10337C10338C10339C10340
 Click-iT EdU Proliferation Assay for Microplates
LabelAmplex UltraRed Reagent, an HRP substrate, is converted into fluorescence signal in presence of HRP.
Ex/Em (nm)568/585
Signal-to-noise ratio
Sample typeOptimized for live cells, the click detection step comes after the fixation.
ReadoutFluorescent signal
Format96-well plate
Cat. No.C10499
 Click-iT EdU Alexa Fluor 488 HCS AssayClick-iT EdU Alexa Fluor 555 HCS AssayClick-iT EdU Alexa Fluor 594 HCS AssayClick-iT EdU Alexa Fluor 647 HCS Assay
Fluorescent labelAlexa Fluor 488 azideAlexa Fluor 555 azideAlexa Fluor 594 azideAlexa Fluor 647 azide
Standard filter setFITCTRITCTexas RedCy5
Ex/Em (nm)495/519555/565590/617650/668
Signal-to-noise ratio
Photostability
BibliographyCitations
ProtocolHCSMicroscopy
Sample typeOptimized for plate-based HCS assays.
MultiplexingCompatible with antibody-fluorophore labeling including standard organic dyes (FITC, Alexa Fluor dyes). Do not use with fluorescent proteins or phalloidin.
ReadoutFluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period
Format10 plates10 plates10 plates10 plates
Cat. No.C10351C10353C10355C10357
 Click-iT EdU Colorimetric IHC Detection Kit
LabelBiotin
BibliographyCitations
ProtocolMicroscopy
Sample typeOptimized for tissue sections
MultiplexingCommonly used secondary tissue stains such as hematoxylin or methyl green can be used to review the cellular context surrounding the proliferation signal.
ReadoutThe peroxidase enzyme and subsequent colorimetric precipitation accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period. The color will be brown.
Format1 kit/50 slides
Cat. No.C10644
 Click-iT Plus EdU Alexa Fluor 488 Imaging KitClick-iT Plus EdU Alexa Fluor 555 Imaging KitClick-iT Plus EdU Alexa Fluor 594 Imaging KitClick-iT Plus EdU Alexa Fluor 647 Imaging Kit
Fluorescent labelAlexa Fluor 488 picolyl azideAlexa Fluor 555 picolyl azideAlexa Fluor 594 picolyl azideAlexa Fluor 647 picolyl azide
Standard filter setFITCTRITCTexas RedCy5
Ex/Em (nm)495/519555/565590/617650
Signal-to-noise ratio
Photostability
BibliographyCitations
ProtocolMicroscopy
Sample typeOptimized for live cells and tissue sections; the click detection step comes after the fixation
MultiplexableCompatible with antibody-fluorophore labeling including standard organic dyes (FITC, Alexa Fluor dyes) and fluorescent proteins such as GFP and mCherry. Do not use with phalloidin.
ReadoutFluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period
Format1 kit/50–250 coverslips1 kit/50–250 coverslips1 kit/50–250 coverslips1 kit/50–250 coverslips
Cat. No.C10637C10638C10639C10640
 Click-iT EdU Alexa Fluor 488 Imaging Kit Click-iT EdU Alexa Fluor 555 Imaging KitClick-iT EdU Alexa Fluor 594 Imaging KitClick-iT EdU Alexa Fluor 647 Imaging Kit
Fluorescent labelAlexa Fluor 488 azideAlexa Fluor 555 axideAlexa Fluor 594 azideAlexa Fluor 647 azide
Standard filter setFITCTRITCTexas RedCy5
Ex/Em (nm)495/519555/565590/617650/668
Signal-to-noise ratio
Photostability
BibliographyCitations
ProtocolMicroscopy
Sample typeOptimized for live cells and tissue sections; the click detection step comes after the fixation
MultiplexableCompatible with antibody-fluorophore labeling including standard organic dyes (FITC, Alexa Fluor dyes). Do not use with fluorescent proteins or phalloidin. 
ReadoutFluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period
Format1 kit/50–250 coverslips1 kit/50–250 coverslips1 kit/50–250 coverslips1 kit/50–250 coverslips
Cat. No.C10337C10338C10339C10340
 Click-iT EdU Proliferation Assay for Microplates
LabelAmplex UltraRed Reagent, an HRP substrate, is converted into fluorescence signal in presence of HRP.
Ex/Em (nm)568/585
Signal-to-noise ratio
Sample typeOptimized for live cells, the click detection step comes after the fixation.
ReadoutFluorescent signal
Format96-well plate
Cat. No.C10499
 Click-iT EdU Alexa Fluor 488 HCS AssayClick-iT EdU Alexa Fluor 555 HCS AssayClick-iT EdU Alexa Fluor 594 HCS AssayClick-iT EdU Alexa Fluor 647 HCS Assay
Fluorescent labelAlexa Fluor 488 azideAlexa Fluor 555 azideAlexa Fluor 594 azideAlexa Fluor 647 azide
Standard filter setFITCTRITCTexas RedCy5
Ex/Em (nm)495/519555/565590/617650/668
Signal-to-noise ratio
Photostability
BibliographyCitations
ProtocolHCSMicroscopy
Sample typeOptimized for plate-based HCS assays.
MultiplexingCompatible with antibody-fluorophore labeling including standard organic dyes (FITC, Alexa Fluor dyes). Do not use with fluorescent proteins or phalloidin.
ReadoutFluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period
Format10 plates10 plates10 plates10 plates
Cat. No.C10351C10353C10355C10357
 Click-iT EdU Colorimetric IHC Detection Kit
LabelBiotin
BibliographyCitations
ProtocolMicroscopy
Sample typeOptimized for tissue sections
MultiplexingCommonly used secondary tissue stains such as hematoxylin or methyl green can be used to review the cellular context surrounding the proliferation signal.
ReadoutThe peroxidase enzyme and subsequent colorimetric precipitation accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period. The color will be brown.
Format1 kit/50 slides
Cat. No.C10644


EdU staining with Click-iT technology, EdU vs BrdU assay

microscopic views of fluorescence staining after BrdU, Click-iT EdU and Click-iT Plus EdU treatment
 Click image to enlarge

Figure 1. BrdU, Click-iT EdU, and Click-iT Plus EdU staining. A375 melanoma cells expressing GFP were imaged for cell proliferation using (A) traditional antibody-based BrdU, (B) Click-iT EdU, and (C) Click-iT Plus EdU. The nucleus is stained blue with Hoechst dye; pink indicates the proliferation signal; green indicates GFP.

Click-iT labeling employs bioorthogonal reactive chemistry with reaction partners, including EdU, that are not endogenously present in biological molecules, cells, tissues, or model organisms. Click chemistry can be used to study cell proliferation through modification of a nucleoside analog that is incorporated during DNA synthesis of actively dividing cells.

Click-iT EdU and Click-iT Plus EdU assays are an alternative to BrdU (5-bromo-2’-deoxyuridine) staining for cell proliferation using a versatile, bioorthogonal, and specific labeling reaction. The Click-iT technology advantage is in the chemistry—small and unique with low-background labeling. The azide-alkyne click reaction is a “spring loaded” reaction that happens quickly and forms a single product with high yield and a strong covalent bond. Given the mild reaction conditions of Click-iT assays, these kits can assess cell proliferation while preserving cell morphology, DNA integrity, antigen binding sites, and fluorescent signal for better DNA staining to examine cell cycles.

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 and extend the time to detection. Such severe treatments can result in inconsistent staining and decreased signal. 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 Click-iT 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, Click-iT EdU cell proliferation kits allow for more consistent results, faster protocols, and better signal to noise compared to an antibody-based BrdU cell proliferation assay.

The simplicity of the click detection method makes the EdU assay a faster alternative to the BrdU assay.

What is BrdU and how does it work?

5-bromo-2’- deoxyuridine (BrdU) is a synthetic analog of thymidine which incorporates into newly synthesized DNA. Proliferating cells labeled with BrdU can be identified using an antibody-based BrdU staining protocol with a flow cytometer or BrdU labeling and detection with a microscope. BrdU staining can be combined with cell surface and/or intracellular targets to help understand cellular function. BrdU and EdU can be used in the same experiment. To minimize cross-reactivity between BrdU and EdU staining, we recommend anti-BrdU clone MoBU-1.

Figure 2. BrdU staining with antibody detection. Denaturation is required for the detection of BrdU that is incorporated into newly synthesized DNA. Acid, heat, and nuclease treatment are options for denaturation. These reagents not only induce unwanted artifacts due to the harsh conditions but also extend the time to detection. Such severe treatments can result in inconsistent staining and decreased staining signal. The protocol shown is for processing rat tissue sections. Choose from optimized kits for BrdU kits for flow cytometry, and anti-BrdU antibodies.

Benefits of using an EdU assay

Unlike assays using BrdU staining, Click-iT EdU assays are not antibody-based and therefore do not require DNA denaturation for detection of the incorporated nucleoside. In addition, for increased utility, the Click-iT Plus EdU assay can be multiplexed with R-phycoerythrin (R-PE) and R-PE tandems, fluorescent proteins (GFP and mCherry), and also with BrdU in a BrdU and EdU double staining experiment. The Click-iT EdU technology has not only been developed into kits for flow cytometry and imaging applications (including HCS), but it also has been adapted for the colorimetric detection of EdU in an immunohistochemistry (IHC) assay. When you compare the methods side-by-side, the benefits of the Click-iT EdU assay and Click-iT Plus EdU assay for cell proliferation are clear.

Figure 3. EdU staining with Click-iT and Click-iT Plus detection. EdU that is incorporated into newly synthesized DNA is detected without the need for DNA denaturation. Simple, easy-to-follow, robust protocols allow for reproducible, bright staining. The Click-iT EdU Assay kits work with most standard fluorophore conjugates. Click-iT Plus EdU Assay kits were designed for maximum multiplex flexibility and are compatible with R-PE (and tandems) and fluorescent proteins such as GFP and mCherry. Protocol shown is for processing rat tissue sections. Optimized kits developed for use in flow cytometry, imaging, microplates, high-content screening, and colorimetric immunohistochemistry applications.

Click-iT vs. Click-iT Plus EdU Cell Proliferation Kits

The Click-iT EdU Cell Proliferation Kits utilize copper-mediated azide-alkyne click cycloaddition for EdU detection. Given that copper can damage proteins and nucleic acids, Click-iT Plus EdU Cell Proliferation Kits were developed and include Invitrogen Alexa Fluor picolyl azides for EdU detection in the presence of copper-sensitive compounds. Picolyl azides react efficiently with chelated copper, so free copper in the click reaction is minimized, protecting against undesired interactions with proteins (e.g., green fluorescent protein (GFP), R-phycoerythrin (R-PE)), nucleic acids (e.g., RNA, oligos), and small molecules. Picolyl azides offer increased sensitivity and faster reaction times than standard azides, and allow multiplexing while retaining the benefits of the standard azide-alkyne click reaction (Table 3). Click-iT Plus assays can determine cell proliferation while preserving cell morphology, DNA integrity, antigen-binding sites, and fluorescent signals for better DNA staining to examine cell cycles.

 Classic Click-iT EdUClick-iT Plus EdU

Detection label functional group

Azide

Picolyl azide

Signal intensity

Bright

As bright or brighter

Reaction rate

Fast

As fast as azide or faster 

Organic dyes such as Alexa Fluor dyes and fluorescein (FITC)

Fluorescent protein including GFP and mCherry compatibility

Not recommended

Phalloidin compatibility

Not recommended

Not recommended

Qdot compatibility

Post click staining

Post click staining

R-PE and R-PE tandem compatibility

Post click staining

PerCP, allophycocyanin (APC) and APC-based tandems (i.e., Alexa Fluor 680-APC)

Polymer dyes including Brilliant Violet dyes compatibility

What is dual-pulse BrdU-EdU staining?

Cell proliferation is traditionally assessed by incubating cells with a single “pulse” of a nucleoside analog that is incorporated into the DNA of actively dividing cells, followed by detection using radioactivity, antibodies, or click chemistry. Many applications benefit from the incorporation of two different analogs at different time points (dual-pulse labeling), which can further distinguish cell proliferation and define cell cycle kinetics. Dual-pulse labeling for cell proliferation is commonly done with BrdU and another analog such as 5-chloro-2´-deoxyuridine (CldU) or 5-iodo-2´- deoxyuridine (IdU), which also require antibodies that have high antigen specificity without cross-reactivity.

The BrdU labeling technique can be combined with click chemistry detection for a simplified method of dual-pulse labeling using EdU when utilizing highly specific anti-BrdU antibodies. For dual-pulse applications, the use of EdU as one of the nucleoside analogs greatly simplifies the procedure, as there is no reactivity between the Click-iT azide detection reagent and the incorporated BrdU. Moreover, several anti-BrdU antibodies, including the MoBU-1 clone, do not cross-react with EdU.

Note: BrdU is preferentially incorporated over EdU, therefore requiring EdU to be administered prior to BrdU in dual-pulse labeling.

Figure 4. Example dual pulse BrdU and EdU staining. Immunofluorescence for EdU (green) and BrdU (red). Data generated by: Gómez-Nicola D, Valle-Argos B, Pallas-Bazarra N, Nieto-Sampedro M. Interleukin-15 regulates proliferation and self-renewal of adult neural stem cells. Mol Biol Cell. 2011 Jun 15;22(12):1960-70. PMID: 21508317

Click-iT EdU sample data

Figure 5. Cell proliferation analysis using the Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit and FxCycle Violet Stain. Jurkat cells were treated with 10 μM EdU for one hour and stained with Invitrogen Alexa Fluor 488 picolyl azide, according to the protocol for the Invitrogen Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit (C10632), followed by staining with Invitrogen FxCycle Violet Stain (F10347). Cells were then analyzed by flow cytometry using either 488 nm excitation (for Click-iT EdU Alexa Fluor 488 dye) or 405 nm excitation (for FxCycle Violet stain). (A) Histogram demonstrating clear separation of cells in S phase (DNA synthesis, including EdU incorporation) and cells in either G2/M or G0/G1. (B) Histogram showing DNA content distribution, with G0/G1 and G2/M phase peaks separated by the S phase distribution using FxCycle Violet stain. (C) Dual parameter Click-iT Plus EdU and FxCycle plot shows co-positive cells that provide a direct measurement of the percentage of cells in S phase.

Multicolor imaging with Click-iT EdU

Figure 6. Cell proliferation multicolor imaging with Click-iT EdU. Muntjac cells pulsed with EdU. Cells were subsequently fixed and permeabilized and labeled DNA detected using Click-iT EdU Alexa Fluor 647 kit (C10085). Tubulin (in blue) is labeled with a mouse primary followed by Alexa Fluor 350 goat anti–mouse IgG antibody. Golgi (in green) is labeled with Alexa Fluor 488 conjugate of lectin HPA (L11271), and peroxisomes (in orange) are labeled with a rabbit primary followed by Alexa Fluor 555 donkey anti–rabbit IgG antibody.

Image of plants cells stained with Click-iT EdU

Figure 7. Click-iT EdU enables detection of DNA synthesis on plant cells without cell wall digestion, currently not possible with BrdU. Detection of DNA synthesis on undigested Medicago sativa (alfalfa) suspension cultures with Click-iT EdU (green). Nuclei counterstained with DAPI (blue). Colocalizing regions appear as cyan. Laser scanning confocal microscopy was performed on Olympus Fluoview FV1000 confocal microscope equipped with 488 nm argon laser (used for Alexa Fluor 488 dye detection) and near-UV 405 nm laser (for DAPI detection). UPLSAPO 60X Oil immersion objective (NA:1.35) was used for image acquisition. Six confocal sections of 1.28 µm/slice were merged onto differential interference contrast image.

Literature and protocols

Protocols for Click-iT kits using imaging applications

Protocols for Click-iT kits using flow cytometry

Experimental design software and webtools

Resources

Tools

Cell viability, proliferation, and cell cycle information—Find educational resources such as application notes, webinars, videos, articles, and more that cover the use of many of our reagents and kits for monitoring cell function.

Support

Cell Analysis Support Center—Find technical information, tips and tricks, and answers to everyday problems.

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