Click-iT EdU Cell Proliferation Assays

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.

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.

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.

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.

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

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.

•  Application note: In vivo use of Click-iT EdU cell proliferation assays
•  Poster: Click-iT EdU Plus for flow cytometry: Enhanced compatibility for multiplexing
•  Poster: Comparison of BrdU method and Click-iT EdU assay in imaging
•  Poster: Effects of the thymidine analogues EdU and BrdU on cell viability and cycle progression
• Publication: Mead TJ, Lefebvre V. Proliferation assays (BrdU and EdU) on skeletal tissue sections. Methods Mol Biol. 2014;1130:233-243. PMID: 24482177
• BioProbes article: Click-iT Labeling Kits Detect Biomolecules Across Multiple Applications
• Click-iT EdU kits FAQs

• Click-iT EdU imaging kits protocol
• Click-iT Plus EdU imaging kits protocol
• Click-iT EdU HCS assays protocol
• BrdU Labeling and detection protocol
• Click-iT EdU labeling in vivo cell proliferation protocol

• BrdU and EdU double-staining protocol

• Click-iT EdU protocol for flow cytometry

•  Click-iT Plus EdU Flow Cytometry Assay Kits

•  BrdU staining protocol for flow cytometry (Invitrogen eBioscience reagents)

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