Single-Pulse and Dual-Pulse Click-iT® EdU Assays

Single-Pulse and Dual-Pulse Cell Proliferation Click-iT® EdU Assays

Click-iT® EdU assays provide a simplified method for detecting nascent DNA synthesis. EdU is an alkyne-containing nucleoside analog of thymidine (5-ethynyl-2’-deoxyuridine), detection of which is based on click chemistry, a copper(I)-catalyzed reaction between an azide and an alkyne. The small size of the detection azide allows the use of mild conditions to access EdU incorporated into the DNA. Even with tissue samples that normally require long incubations, a Click-iT® EdU assay is complete in less than 90 minutes without the need for secondary detection or signal amplification. Either alone or with the more conventional nucleoside analog bromo-2’-deoxyuridine (BrdU), Click-iT® EdU assays allow researchers to follow temporal changes in cell proliferation.

Single-Pulse Cell Proliferation Studies

Despite its shortcomings, until the introduction of Click-iT® EdU assays, the method of choice to label dividing cells has been BrdU. Early work labeling adult dentate gyrus neurons in rats showed that 200–300 mg/kg BrdU was needed to label all of the actively proliferating neurons; BrdU labeling at lower concentrations of 50 and 100 mg/kg detected only 40% and 60% of the S-phase cells, respectively [1]. To label neurons in the hippocampal subgranular zone in mice, 150 mg/kg BrdU is required; 25–50 mg/kg BrdU results in very faint, nearly undetectable labeling [2]. In a recent publication comparing EdU to BrdU to label adult neurons in mice using a dual-pulse technique to validate the usage and dosage of EdU in a single-pulse application, 200 mg/kg EdU labeled the same neurons as an equivalent amount of BrdU [3]; unlike previous work using BrdU, in which lowering the nucleoside concentration resulted in faint or inefficient labeling, when mouse dentate gyrus neurons were labeled with only 50 mg/kg EdU, near-saturating levels of fluorescence corresponding to cells in S-phase were detected.

In applications where only a single pulse is required, EdU provides not only a greatly truncated protocol compared to BrdU by eliminating the DNA denaturation step, but also an extremely bright signal without the incubations required by secondary detection methods for signal amplification. A comparison of rat intestine signal intensity when pulsed with EdU or BrdU showed very bright click-label EdU signal after only a 1-hour incubation, compared to BrdU antibody labeling that required an overnight incubation (Figure 1). A titration study in U2OS cells comparing equal exposures of cells treated with a pulse of EdU or BrdU showed that cells click-labeled with EdU had brighter signal than cells labeled with BrdU and detected using a directly labeled antibody (Figure 2).

Figure 1. Comparison of signal strength in tissues labeled with EdU and BrdU. Sectioned formaldehyde-fixed, paraffin-embedded tissue was prepared from rats injected intraperitoneally with EdU (Right panel) or BrdU (Left panel). Proliferating cells within the intestinal villi that incorporated the nucleoside are pink. EdU was detected using the reagents in the Click-iT® EdU Alexa Fluor® 647 Imaging Kit. BrdU-labeled tissue was denatured with HCl, neutralized, and blocked prior to incubating with mouse anti-BrdU antibody overnight. After washing in blocking buffer, the tissue was incubated with goat anti-mouse Alexa Fluor® 647 conjugate. Nuclei in both samples were counterstained with Hoechst 33342 (gray). Images were acquired with a 20x objective on a Nikon Eclipse 800 epifluorescence microscope using a 5 msec exposure for EdU and 2,000 msec for BrdU, demonstrating the greater brightness of the EdU signal.

Figure 2. Comparison of signal strength of BrdU and EdU labeling in cultured cells. U2OS cells were grown in 96-well plates, pulsed with 3–10 µM BrdU or EdU for 2 hours, then formaldehyde-fixed and permeabilized, HCl-treated to denature DNA (for BrdU samples), and neutralized. Cells were then labeled with Click-iT® EdU Alexa Fluor® 488 reaction cocktail for 1 hr to detect EdU, or incubated overnight with the anti-BrdU Alexa Fluor® 488 conjugate to detect BrdU. Data were collected on a Cellomics ArrayScan® VTI imaging platform and are expressed as the average fluorescence intensity from nuclear regions.

Dual-Pulse Cell Proliferation Studies

Historically, double-labeling of tissue with halogenated thymidine analogs has required significant effort to demonstrate the validity of the approach. As a result, several protocols have been developed. Not only do the analogs, including BrdU, 5-chloro-2’-deoxyuridine (CldU), and 5-iodo-2’-deoxyuridine (IdU), need to be equivalently incorporated into the tissue to result in equal brightness, but the antibodies used to detect them must have defined specificity and cross-reactivity. One approach has been to take advantage of the cross-reactivity of BrdU antibodies. Using a mouse BrdU antibody with cross-reactivity to IdU and a rat BrdU antibody without cross-reactivity to IdU to detect IdU/BrdU dual-pulse–labeled mouse tissue resulted in single-color labeling of cells that incorporated IdU, and two-color labeling of BrdU- or dual-pulsed cells [4]. In a slightly different approach, tissue dual-pulsed with CldU and IdU was detected with two antibodies: one with BrdU and IdU reactivity, and the other with BrdU and CldU reactivity [5].

Using EdU as one of the analogs makes dual-pulse labeling much easier, as there is no reactivity between the click dye azide and the incorporated BrdU. It is still important to establish that the BrdU antibody doesn’t cross-react with EdU. Several BrdU antibody clones, including MoBU-1, do not cross-react with EdU.

Together, BrdU and EdU have allowed temporal characterization of S-phase cells to investigate the relationship between cell cycle length and neurogenesis [3]. To answer questions about the length of the cell cycle during embryonic development, Lange et al. [6] used BrdU/EdU dual-pulse labeling to identify cells entering S-phase. When both pulses generated punctate signals within a cell, that cell had just entered S-phase. Homogeneous signal from both pulses was interpreted as a cell in mid-S-phase, while a punctate signal from the first pulse (BrdU) but no signal from the second pulse (EdU) was interpreted as a cell exiting S-phase.

A Stronger Pulse

Click-iT® EdU assays represent a breakthrough in detecting nascent DNA synthesis, made possible by the orthogonal detection modality of click chemistry. These assays are a powerful tool for identifying temporal changes in cell proliferation. For more information on Click-iT® EdU assays and other reagents using click chemistry, visit Click-iT® EdU Cell Proliferation Assays.