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The Invitrogen Click-iT EdU cell proliferation assays combine EdU labeling with powerful click chemistry to provide a superior alternative to traditional BrdU staining methods for detecting and quantitating newly synthesized DNA.
Cell Proliferation Assay Protocols Cell Cycle & Proliferation Pathways
The simplicity of the click detection method makes the EdU assay a faster, friendlier alternative to the 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.
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.
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.
Ideal for maximum multiplexability in your flow cytometry experiments. Compatible with R-PE, R-PE tandems, and fluorescent proteins such as GFP and mCherry.
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 |
| ||||||
Multiplexable | Maximum multiplexability
| ||||||
Readout | Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period. | ||||||
Fluorescent 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 |
Limited multiplexability. Not compatible with R-PE, R-PE tandems and fluorescent proteins such as GFP and mCherry.
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 |
| ||||
Multiplexable | Limited multiplexability
| ||||
Readout | Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period. | ||||
Fluorescent label | Pacific Blue azide | Alexa Fluor 488 azide | Alexa Fluor 647 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 |
Ideal for maximum multiplexability in your flow cytometry experiments. Compatible with R-PE, R-PE tandems, and fluorescent proteins such as GFP and mCherry.
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 |
| ||||||
Multiplexable | Maximum multiplexability
| ||||||
Readout | Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period. | ||||||
Fluorescent 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 |
Limited multiplexability. Not compatible with R-PE, R-PE tandems and fluorescent proteins such as GFP and mCherry.
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 |
| ||||
Multiplexable | Limited multiplexability
| ||||
Readout | Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period. | ||||
Fluorescent label | Pacific Blue azide | Alexa Fluor 488 azide | Alexa Fluor 647 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 |
Ideal for maximum multiplexability in imaging experiments. Compatible with fluorescent proteins such as GFP and mCherry.
Click-iT Plus EdU Alexa Fluor 488 Imaging Kit | Click-iT Plus EdU Alexa Fluor 555 Imaging Kit | Click-iT Plus EdU Alexa Fluor 594 Imaging Kit | Click-iT Plus EdU Alexa Fluor 647 Imaging Kit | |
---|---|---|---|---|
Basis of assay |
| |||
Multiplexable | Maximum multiplexability
| |||
Readout |
| |||
Fluorescent label | Alexa Fluor 488 picolyl azide | Alexa Fluor 555 picolyl azide | Alexa Fluor 594 picolyl azide | Alexa Fluor 647 picolyl azide |
Standard filter set | FITC | TRITC | Texas Red | Cy5 |
Ex/Em (nm) | 495/519 | 555/565 | 590/617 | 650 |
Signal-to-noise ratio | ![]() | ![]() | ![]() | ![]() |
Photostability | ![]() | ![]() | ![]() | ![]() |
Bibliography | Citations | |||
Protocol | Microscopy | |||
Sample type | Optimized for live cells and tissue sections; the click detection step comes after fixation. | |||
Format | 1 kit/50–250 coverslips | 1 kit/50–250 coverslips | 1 kit/50–250 coverslips | 1 kit/50–250 coverslips |
Cat. No. | C10637 | C10638 | C10639 | C10640 |
Limited multiplexability in imaging experiments. Not compatible with fluorescent proteins.
Click-iT EdU Alexa Fluor 488 Imaging Kit | Click-iT EdU Alexa Fluor 555 Imaging Kit | Click-iT EdU Alexa Fluor 594 Imaging Kit | Click-iT EdU Alexa Fluor 647 Imaging Kit | |
---|---|---|---|---|
Basis of assay |
| |||
Multiplexable |
| |||
Readout |
| |||
Fluorescent label | Alexa Fluor 488 | Alexa Fluor 555 | Alexa Fluor 594 | Alexa Fluor 647 |
Standard filter set | FITC | TRITC | Texas Red | Cy5 |
Ex/Em (nm) | 495/519 | 555/565 | 590/617 | 650/668 |
Signal-to-noise ratio | ![]() | ![]() | ![]() | ![]() |
Photostability | ![]() | ![]() | ![]() | ![]() |
Bibliography | Citations | |||
Protocol | Microscopy | |||
Sample type | Optimized for live cells; the click detection step comes after fixation. | |||
Format | 1 kit/50–250 coverslips | 1 kit/50–250 coverslips | 1 kit/50–250 coverslips | 1 kit/50–250 coverslips |
Cat. No. | C10337 | C10338 | C10339 | C10340 |
Click-iT EdU kit designed and optimized for microplate readers.
Click-iT EdU Proliferation Assay for Microplates | |
---|---|
Basis of assay |
|
Readout | Fluorescence of highly fluorescent product from reaction of Amplex UltraRed with HRP |
Ex/Em (nm) | 568/585 |
Signal-to-noise ratio | ![]() |
Protocol | Plate reader |
Sample type | Optimized for live cells, the click detection step comes after the fixation. |
Format | 96-well plate |
Cat. No. | C10499 |
Click-iT EdU kits designed and optimized for high-content screen applications.
Click-iT EdU Alexa Fluor 488 HCS Assay | Click-iT EdU Alexa Fluor 555 HCS Assay | Click-iT EdU Alexa Fluor 594 HCS Assay | Click-iT EdU Alexa Fluor 647 HCS Assay | |
---|---|---|---|---|
Basis of assay |
| |||
Readout |
| |||
Fluorescent label | Alexa Fluor 488 | Alexa Fluor 555 | Alexa Fluor 594 | Alexa Fluor 647 |
Standard filter set | FITC | TRITC | Texas Red | Cy5 |
Ex/Em (nm) | 495/519 | 555/565 | 590/617 | 650/668 |
Signal-to-noise ratio | ![]() | ![]() | ![]() | ![]() |
Photostability | ![]() | ![]() | ![]() | ![]() |
Bibliography | Citations | |||
Protocol | HCS, Microscopy | |||
Multiplexing | The mild click chemistry protocol does not destroy cellular epitopes (unlike BrdU protocols) and is compatible with antibody labeling and common cell- or tissue-staining methods. | |||
Sample type | Optimized for plate-based HCS assays. Reagents can be added directly to culture medium. | |||
Format | 10 plates | 10 plates | 10 plates | 10 plates |
Cat. No. | C10351 | C10353 | C10355 | C10357 |
The Click-iT EdU Colorimetric IHC Detection Kit is perfect for tissues with high background fluorescence; the proliferation signal that can be archived for future analysis.
Click-iT EdU Colorimetric IHC Detection Kit | |
---|---|
Basis of assay |
|
Readout |
|
Label | Colorimetric detection |
Platform | Light microscopy |
Bibliography | Citations |
Protocol | Microscopy |
Multiplexing | Commonly used secondary tissue stains such as hematoxylin or methyl green can be used to review the cellular context surrounding the proliferation signal. |
Sample type | Optimized for tissue sections |
Format | 1 kit/50 slides |
Cat. No. | C10644 |
Ideal for maximum multiplexability in imaging experiments. Compatible with fluorescent proteins such as GFP and mCherry.
Click-iT Plus EdU Alexa Fluor 488 Imaging Kit | Click-iT Plus EdU Alexa Fluor 555 Imaging Kit | Click-iT Plus EdU Alexa Fluor 594 Imaging Kit | Click-iT Plus EdU Alexa Fluor 647 Imaging Kit | |
---|---|---|---|---|
Basis of assay |
| |||
Multiplexable | Maximum multiplexability
| |||
Readout |
| |||
Fluorescent label | Alexa Fluor 488 picolyl azide | Alexa Fluor 555 picolyl azide | Alexa Fluor 594 picolyl azide | Alexa Fluor 647 picolyl azide |
Standard filter set | FITC | TRITC | Texas Red | Cy5 |
Ex/Em (nm) | 495/519 | 555/565 | 590/617 | 650 |
Signal-to-noise ratio | ![]() | ![]() | ![]() | ![]() |
Photostability | ![]() | ![]() | ![]() | ![]() |
Bibliography | Citations | |||
Protocol | Microscopy | |||
Sample type | Optimized for live cells and tissue sections; the click detection step comes after fixation. | |||
Format | 1 kit/50–250 coverslips | 1 kit/50–250 coverslips | 1 kit/50–250 coverslips | 1 kit/50–250 coverslips |
Cat. No. | C10637 | C10638 | C10639 | C10640 |
Limited multiplexability in imaging experiments. Not compatible with fluorescent proteins.
Click-iT EdU Alexa Fluor 488 Imaging Kit | Click-iT EdU Alexa Fluor 555 Imaging Kit | Click-iT EdU Alexa Fluor 594 Imaging Kit | Click-iT EdU Alexa Fluor 647 Imaging Kit | |
---|---|---|---|---|
Basis of assay |
| |||
Multiplexable |
| |||
Readout |
| |||
Fluorescent label | Alexa Fluor 488 | Alexa Fluor 555 | Alexa Fluor 594 | Alexa Fluor 647 |
Standard filter set | FITC | TRITC | Texas Red | Cy5 |
Ex/Em (nm) | 495/519 | 555/565 | 590/617 | 650/668 |
Signal-to-noise ratio | ![]() | ![]() | ![]() | ![]() |
Photostability | ![]() | ![]() | ![]() | ![]() |
Bibliography | Citations | |||
Protocol | Microscopy | |||
Sample type | Optimized for live cells; the click detection step comes after fixation. | |||
Format | 1 kit/50–250 coverslips | 1 kit/50–250 coverslips | 1 kit/50–250 coverslips | 1 kit/50–250 coverslips |
Cat. No. | C10337 | C10338 | C10339 | C10340 |
Click-iT EdU kit designed and optimized for microplate readers.
Click-iT EdU Proliferation Assay for Microplates | |
---|---|
Basis of assay |
|
Readout | Fluorescence of highly fluorescent product from reaction of Amplex UltraRed with HRP |
Ex/Em (nm) | 568/585 |
Signal-to-noise ratio | ![]() |
Protocol | Plate reader |
Sample type | Optimized for live cells, the click detection step comes after the fixation. |
Format | 96-well plate |
Cat. No. | C10499 |
Click-iT EdU kits designed and optimized for high-content screen applications.
Click-iT EdU Alexa Fluor 488 HCS Assay | Click-iT EdU Alexa Fluor 555 HCS Assay | Click-iT EdU Alexa Fluor 594 HCS Assay | Click-iT EdU Alexa Fluor 647 HCS Assay | |
---|---|---|---|---|
Basis of assay |
| |||
Readout |
| |||
Fluorescent label | Alexa Fluor 488 | Alexa Fluor 555 | Alexa Fluor 594 | Alexa Fluor 647 |
Standard filter set | FITC | TRITC | Texas Red | Cy5 |
Ex/Em (nm) | 495/519 | 555/565 | 590/617 | 650/668 |
Signal-to-noise ratio | ![]() | ![]() | ![]() | ![]() |
Photostability | ![]() | ![]() | ![]() | ![]() |
Bibliography | Citations | |||
Protocol | HCS, Microscopy | |||
Multiplexing | The mild click chemistry protocol does not destroy cellular epitopes (unlike BrdU protocols) and is compatible with antibody labeling and common cell- or tissue-staining methods. | |||
Sample type | Optimized for plate-based HCS assays. Reagents can be added directly to culture medium. | |||
Format | 10 plates | 10 plates | 10 plates | 10 plates |
Cat. No. | C10351 | C10353 | C10355 | C10357 |
The Click-iT EdU Colorimetric IHC Detection Kit is perfect for tissues with high background fluorescence; the proliferation signal that can be archived for future analysis.
Click-iT EdU Colorimetric IHC Detection Kit | |
---|---|
Basis of assay |
|
Readout |
|
Label | Colorimetric detection |
Platform | Light microscopy |
Bibliography | Citations |
Protocol | Microscopy |
Multiplexing | Commonly used secondary tissue stains such as hematoxylin or methyl green can be used to review the cellular context surrounding the proliferation signal. |
Sample type | Optimized for tissue sections |
Format | 1 kit/50 slides |
Cat. No. | C10644 |
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).
Figure 3. 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 Invitrogen Alexa Fluor 488 picolyl azide, according to the protocol for the Invitrogen Click-iT Plus EdU Alexa Fluor 488 Flow Cytometry Assay Kit, followed by staining with Invitrogen 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.
Figure 4. 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 Invitrogen FxCycle Far Red stain for DNA content; cells that are co-positive are in S phase.
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.
Figure 5. Cell proliferation assays using A375 melanoma cells expressing GFP comparing traditional antibody-based BrdU (A), Click-iT EdU (B), and Click-iT Plus EdU (C). The nucleus is stained blue with Hoechst, the pink is the proliferation signal, and the green is from the GFP.
Figure 6. 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-Cy7 fluorescence.
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