Click-iT® Plus EdU for Flow Cytometry

Replace your cumbersome BrdU assays

The Molecular Probes™ Click-iT™ EdU cell proliferation assay utilizes the power of click chemistry to provide a superior alternative to traditional BrdU methods for detecting and quantitating newly synthesized DNA. When the modified nucleoside, EdU (5-ethynyl-2′-deoxyuridine), is incorporated during DNA synthesis, it can be detected in a quick “click chemistry” reaction with minimal disruption to the cell.

Click-iT EdU—click chemistry simplicity makes it the faster, friendlier alternative to BrdU. Unlike assays using bromodeoxyuridine (BrdU), 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, Click-iT EdU can be multiplexed with fluorescent proteins like R-PE, R-PE tandems, and GFP. When you compare the methods side-by-side, the benefits of Click-iT EdU and Click-iT Plus EdU for cell proliferation are clear. (Table 1).

“The Click-iT EdU technology has almost entirely replaced older BrdU methods for detecting cell proliferation by flow cytometry; it is simpler, fast and far more gentle to cells than old techniques. The Click-iT Plus kits have provided additional enhancements to this now-standard technology, with improved sensitivity and compatible with PE and fluorescent proteins, allowing easy multicolor analysis of cell division. We now teach the Click-iT EdU Plus method in our flow cytometry workshops as the new standard technique for measuring cell proliferation.”

—Bill Telford, PhD, National Cancer Institute, NIH

The Click-iT EdU technology is:

  • Fast—detection in as little as 60 minutes.
  • Accurate—no artifacts arising from the use of antibodies and denaturation steps.
  • Streamlined—simple six-step protocol.

Additionally the Click-iT Plus EdU kits are:

  • Multiplexable—compatible with R-PE (and tandems) and fluorescent proteins.

Table 1. Comparison of Click-iT EdU, Click-it Plus EdU, and BrdU assays.

  Click-iT EdU Kits Click-iT Plus EdU Kits BrdU
Multiplexable With most standard fluorophore conjugates (excluding FPs and tandems)
With all standard fluorophore conjugates (including GFP and other FPs and sensitive tandems)
Variable
Reagents required for basic experiment
Time to results
(labeling+detection)
Typically <90 min
5 hours to overnight
Number of steps
6
(No need for multiple permeabilization steps,
no need for DNase treatment)
~10
DNA denaturation
None
Required

Click-iT Edu Assay Kits selection guides

  Click-iT Plus EdU Alexa Fluor 350 Flow Cytometry Kit Click-iT Plus EdU Pacific Blue™ Flow Cytometry Kit Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Kit Click-iT Plus EdU Alexa Fluor 594 Flow Cytometry Kit Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Kit
Basis of assay A replacement for traditional BrdU assays, the modified thymidine analog EdU is incorporated into newly synthesized DNA in proliferating cells. It labels the cells with a bright, photostable Alexa Fluor dye in a fast, highly specific click reaction.
Multiplexable The Click-iT EdU Plus kits provide maximum multiplexability. The mild click chemistry protocol does not destroy cellular epitopes (unlike BrdU protocols) and is compatible with standard antibody labels (small organic dyes such as FITC or the Alexa Fluor dyes and also sensitive tandems such as R-PE-Cy®5 or APC-Cy®7), fluorescent proteins, and common cell staining methods.
Readout Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period.
Fluoresent label Alexa Fluor 350 picolyl azide Pacific Blue picolyl azide Alexa Fluor 488 picolyl azide Alexa Fluor 594 picolyl azide Alexa Fluor 647 picolyl azide
Laser (nm) UV 405 488 532 or 561 633/635
Ex/Em (nm) 350/438 410/455 495/519 532 or 561/615 650/668
Sample type Optimized for live cells, the click detection step comes after the fixation.
Bibliography
Format 50 assays 50 assays 50 assays 100 assays 50 assays 50 assays 100 assays
Cat. No. C10645 C10636 C10632 C10633 C10646 C10634 C10635
  Click-iT EdU Pacific Blue Flow Cytometry Kit Click-iT EdU Alexa Fluor 488 Flow Cytometry Kit Click-iT EdU Alexa Fluor 647 Flow Cytometry Kit
Basis of assay The standard Click-iT EdU kits have limited compatibility; they are compatible with organic dye labels (such as FITC, or the Alexa Fluor dyes), but they are not compatible with fluorescent proteins or R-phycoerythrin (R-PE) and R-PE based tandems (i.e., R-PE-Cy®5 conjugates).
Multiplexable The standard Click-iT EdU kits have limited compatibility; they are compatible with organic dye labels (such as FITC, or the Alexa Fluor dyes), but they are not compatible with fluorescent proteins or R-phycoerythrin (R-PE) and R-PE based tandems (i.e., R-PE-Cy®5 conjugates).
For compatibility with fluorescent proteins and sensitive APC- and R-PE-based tandems, we recommend use of the Click-iT EdU Plus flow cytometry kits.
Readout Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period.
Fluoresent label Pacific Blue picolyl azide Alexa Fluor 488 picolyl azide Alexa Fluor 647 picolyl azide
Laser 405 nm 488 nm 633/635 nm
Ex/Em (nm) 410/455 495/519 650/668
Sample type Optimized for live cells, the click detection step comes after the fixation.
Bibliography
Format 50 assays 50 assays 100 assays 50 assays 100 assays
Cat. No. C10418 C10424 C10420 C10425 C10419

The science behind Click-iT EdU Kits for flow cytometry

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 1 and 2).

 

Figure 1. 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 Alexa Fluor 488 picolyl azide, according to the protocol for the Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit, followed by staining with FxCycle Violet Stain. 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.
 
Click-iT® Plus for Flow Cytometry
Figure 2. Alexa Fluor™ 488 Click-iT™ Plus EdU labeling with mCherry fluorescent protein and FxCycle™ Far Red stain. mCherry-expressing A549 cells were treated with 10 µM EdU for 2 hours and detected according to the recommended staining protocol. Panel A shows a histogram of cells labeled with Alexa Fluor 488 picolyl azide, identifying cells in S-phase. Panels B and C show mCherry fluorescence from cells that were labeled with Click-iT Plus EdU Alexa Fluor 488 picolyl azide in the presence of copper (Panel B) and in the absence of copper (Panel C), demonstrating that the mCherry fluorescence is preserved in the click reaction. Panel D shows a dual parameter plot of Click-iT Plus EdU Alexa Fluor 488 and FxCycle Far Red stain for DNA content; cells that are co-positive are in S-phase.

Multiplex capabilites with Click-iT Plus EdU reagents

The Click-iT Plus formulation provides increased multiplexability compared to the original Click-iT EdU flow cytometry 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.

Multiplex with:

  • Most standard antibody conjugates available
  • Sensitive R-PE fluorophores such as R-PE-Cy®7
  • GFP, mCherry and other fluorescent proteins
  • Cell surface and intracellular markers
  • Cell cycle dyes
 


Figure 3. Results from immunophenotyping experiment to evaluate CD3 and DNA strand breaks in human T cells.
Dual parameter plot of Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit and Hu CD3 PE-Cy®7 fluorescence.