Single-Pulse Cell Proliferation Studies
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
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 . To answer questions about the length of the cell cycle during embryonic development, Lange et al.  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
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- Learn More about Click-iT® EdU Cell Proliferation Assays