Click-iT® EdU Assay Kits for Flow Cytometry

Click-iT® EdU Flow Cytometry Assay Kits completely eliminate the DNA denaturation step required by BrdU assays (typically using HCl, heat, or digestion with DNase)—steps that not only disrupt dsDNA integrity and thus can affect nuclear counterstaining, but can also destroy cell morphology and antigen recognition sites. The Click-iT® EdU Flow Cytometry Assay Kits provide a simplified, more robust assay for analyzing DNA replication in proliferating cells compared to traditional BrdU methods.

BrdU assays are dependent upon thymidine analog detection by an anti-BrdU antibody, leading to significant lot-to-lot variability due to the specificity of the antibody. This can result in variability from experiment to experiment. In contrast, Click-iT® EdU Kits for flow cytometry have much less variability from one experiment to the next because of the azide-alkyne reaction.

In just five steps you’ll be ready to analyze your cell proliferation data.

1. Treat cells with EdU
2. Fix and permeabilize cells
3. Detect S-phase cells with Click-iT® detection cocktail for 30 minutes
4. Wash once
5. Analyze

The new assay kits contain 50% fewer components than the original kits. The optimized permeabilization reagent and streamlined protocol make the new Click-iT® EdU Flow Cytometry Assay Kits simpler than ever.

The Click-iT® EdU 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 Pacific Blue™ dye–labeled azide, forming a stable covalent bond. The small size of the azide reagents allows efficient access to the DNA without the need for harsh cell treatment, thus simplifying the assay considerably, yet generating the same results (Figures 1 and 2). Click-iT® EdU labeling is compatible with most fixation protocols.

click to enlarge		Figure 1. Analysis of cell proliferation using the Click-iT® EdU Pacific Blue™ Flow Cytometry Assay Kit. Jurkat cells were treated with 10 µM EdU for 2 hr and detected according to the staining protocol. Cells were labeled with Pacific Blue™ azide and analyzed on the Attune® Acoustic Focusing Cytometer using 405 nm excitation with a 450/50 bandpass.
click to enlarge		Figure 2. Flow cytometric analysis of cell proliferation using Click-iT® technology. Jurkat cells were treated with 10 µM EdU for 2 hr, then fixed and permeabilized, stained with the click reaction, washed, and counterstained for cell cycle analysis using SYTOX® AADvanced™ stain. Cells were analyzed using the Attune® Acoustic Focusing Cytometer using 405 nm and 488 nm excitation and 450/50 nm bandpass and 675/20 nm bandpass filters.