Click-iT EdU Assays—A Superior BrdU Alternative

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

BrdU assay

In the traditional BrdU assay, cells proliferating in the window of time that the thymidine nucleoside analog BrdU is present, will incorporate BrdU into newly synthesized DNA. These proliferating cells can be identified using an antibody-based BrdU staining protocol that is amenable to staining with other cell surface and/or intracellular targets of interest in flow cytometry. In microscopy experiments, BrdU labeling and detection can be combined with secondary antibodies.
 

Figure 1. BrdU staining with antibody detection. Denaturation is required for 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.
 

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 (Figures 1 and 2), the benefits of the Click-iT EdU assay and Invitrogen Click-iT Plus EdU assay for cell proliferation are clear.

• 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

Figure 2. 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 technology advantage is in the chemistry—small, unique, and low-background labeling and detection moieties that react specifically and covalently with one another. 5-ethynyl-2´-deoxyuridine (EdU) is a nucleoside analog containing an alkyne. In a copper-catalyzed reaction, the alkyne reacts with a dye-labeled azide, forming a stable covalent bond. The small size of the azide reagent allows efficient access to the DNA without the need for harsh cell treatment. This simplifies the assay considerably, and the results you achieve are similar to (or better than) those typically achieved using BrdU (Figures 3 and 4).

The copper concentrations typically used in traditional click chemistry reactions can affect fluorophores such as GFP, mCherry, R-PE, and R-PE tandem dyes. The Click-iT Plus formulation may be effectively employed in a low-copper reaction, enabling increased multiplexability compared to the original Click-iT EdU assays. Click-iT Plus EdU assays can be used in conjunction with R-PE and R-PE tandems, as well as fluorescent proteins such as GFP and mCherry, without loss of the accuracy or speed of the original Click-iT EdU assay. The low-copper reaction with Click-iT Plus EdU results in bright signals and retains the fluorescent signal from GFP (Figure 5). In addition, detection of EdU with the Click-iT Plus Alexa Fluor 488 picolyl azide was compatible with PE-Cy7 fluorescence in a flow cytometric assay for cell proliferation (Figure 6). Select from Click-iT Plus EdU kits for flow cytometry or Click-iT Plus EdU assays for imaging applications.

Multiplex with:

• Most standard antibody conjugates available
• Sensitive R-PE fluorophores such as R-PE-Cy7
• GFP, mCherry, and other fluorescent proteins
• Cell surface and intracellular markers
• Cell cycle dyes

• Application note: In vivo use of Click-iT EdU cell proliferation assays
• Poster: Click-iT EdU Plus for flow cytometry: Enhanced compatibility for multiplexing