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The Invitrogen Click-iT EdU cell proliferation assays combine EdU (5-ethynyl 2´-deoxyuridine) labeling with click chemistry to provide an alternative to traditional BrdU staining methods for detecting and quantitating newly synthesized DNA. Click-iT EdU assays simplify the experimental process while providing flexibility to use a variety of fluorescent conjugates. Click chemistry is important in numerous applications, including flow cytometry, immunohistochemistry, high content screening and fluorescent imaging techniques.
Benefits include:
Learn how to use Click-iT EdU kits to measure or observe cell proliferation in animal models. Includes protocols and references.
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 | |||
---|---|---|---|---|---|---|---|
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 | |||||||
Multiplexable | Can be co-stained with standard organic dyes (FITC, Alexa Fluor dyes, etc.); R-PE, R-PE tandems (such as R-PE-Cy5 or R-PE-Cy7); Fluorescent proteins (such as GFP and mCherry) | ||||||
Readout | Histogram may show separation of cells in S phase (DNA synthesis, including EdU incorporation) | ||||||
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 | |||
---|---|---|---|---|---|
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 | |||||
Multiplexable | Limited multiplexability; can be co-stained with standard organic dyes (FITC, Alexa Fluor dyes, etc.). Not compatible with R-PE, R-PE tandems (such as R-PE-Cy5 or R-PE-Cy7); fluorescent proteins | ||||
Readout | Histogram may show separation of cells in S phase (DNA synthesis, including EdU incorporation) | ||||
Format | 50 assays | 50 assays | 100 assays | 50 assays | 100 assays |
Cat. No. | C10418 | C10424 | C10420 | C10425 | C10419 |
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 | |||
---|---|---|---|---|---|---|---|
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 | |||||||
Multiplexable | Can be co-stained with standard organic dyes (FITC, Alexa Fluor dyes, etc.); R-PE, R-PE tandems (such as R-PE-Cy5 or R-PE-Cy7); Fluorescent proteins (such as GFP and mCherry) | ||||||
Readout | Histogram may show separation of cells in S phase (DNA synthesis, including EdU incorporation) | ||||||
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 | |||
---|---|---|---|---|---|
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 | |||||
Multiplexable | Limited multiplexability; can be co-stained with standard organic dyes (FITC, Alexa Fluor dyes, etc.). Not compatible with R-PE, R-PE tandems (such as R-PE-Cy5 or R-PE-Cy7); fluorescent proteins | ||||
Readout | Histogram may show separation of cells in S phase (DNA synthesis, including EdU incorporation) | ||||
Format | 50 assays | 50 assays | 100 assays | 50 assays | 100 assays |
Cat. No. | C10418 | C10424 | C10420 | C10425 | C10419 |
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 | |
---|---|---|---|---|
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 the fixation | |||
Multiplexable | Compatible with antibody-fluorophore labeling including standard organic dyes (FITC, Alexa Fluor dyes) and fluorescent proteins such as GFP and mCherry. Do not use with phalloidin. | |||
Readout | Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period | |||
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 |
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 | |
---|---|---|---|---|
Fluorescent label | Alexa Fluor 488 azide | Alexa Fluor 555 axide | Alexa Fluor 594 azide | Alexa Fluor 647 azide |
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 and tissue sections; the click detection step comes after the fixation | |||
Multiplexable | Compatible with antibody-fluorophore labeling including standard organic dyes (FITC, Alexa Fluor dyes). Do not use with fluorescent proteins or phalloidin. | |||
Readout | Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period | |||
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 Proliferation Assay for Microplates | |
---|---|
Label | Amplex UltraRed Reagent, an HRP substrate, is converted into fluorescence signal in presence of HRP. |
Ex/Em (nm) | 568/585 |
Signal-to-noise ratio | ![]() |
Sample type | Optimized for live cells, the click detection step comes after the fixation. |
Readout | Fluorescent signal |
Format | 96-well plate |
Cat. No. | C10499 |
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 | |
---|---|---|---|---|
Fluorescent label | Alexa Fluor 488 azide | Alexa Fluor 555 azide | Alexa Fluor 594 azide | Alexa Fluor 647 azide |
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 | |||
Sample type | Optimized for plate-based HCS assays. | |||
Multiplexing | Compatible with antibody-fluorophore labeling including standard organic dyes (FITC, Alexa Fluor dyes). Do not use with fluorescent proteins or phalloidin. | |||
Readout | Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period | |||
Format | 10 plates | 10 plates | 10 plates | 10 plates |
Cat. No. | C10351 | C10353 | C10355 | C10357 |
Click-iT EdU Colorimetric IHC Detection Kit | |
---|---|
Label | Biotin |
Bibliography | Citations |
Protocol | Microscopy |
Sample type | Optimized for tissue sections |
Multiplexing | Commonly used secondary tissue stains such as hematoxylin or methyl green can be used to review the cellular context surrounding the proliferation signal. |
Readout | The peroxidase enzyme and subsequent colorimetric precipitation accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period. The color will be brown. |
Format | 1 kit/50 slides |
Cat. No. | C10644 |
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 | |
---|---|---|---|---|
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 the fixation | |||
Multiplexable | Compatible with antibody-fluorophore labeling including standard organic dyes (FITC, Alexa Fluor dyes) and fluorescent proteins such as GFP and mCherry. Do not use with phalloidin. | |||
Readout | Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period | |||
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 |
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 | |
---|---|---|---|---|
Fluorescent label | Alexa Fluor 488 azide | Alexa Fluor 555 axide | Alexa Fluor 594 azide | Alexa Fluor 647 azide |
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 and tissue sections; the click detection step comes after the fixation | |||
Multiplexable | Compatible with antibody-fluorophore labeling including standard organic dyes (FITC, Alexa Fluor dyes). Do not use with fluorescent proteins or phalloidin. | |||
Readout | Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period | |||
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 Proliferation Assay for Microplates | |
---|---|
Label | Amplex UltraRed Reagent, an HRP substrate, is converted into fluorescence signal in presence of HRP. |
Ex/Em (nm) | 568/585 |
Signal-to-noise ratio | ![]() |
Sample type | Optimized for live cells, the click detection step comes after the fixation. |
Readout | Fluorescent signal |
Format | 96-well plate |
Cat. No. | C10499 |
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 | |
---|---|---|---|---|
Fluorescent label | Alexa Fluor 488 azide | Alexa Fluor 555 azide | Alexa Fluor 594 azide | Alexa Fluor 647 azide |
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 | |||
Sample type | Optimized for plate-based HCS assays. | |||
Multiplexing | Compatible with antibody-fluorophore labeling including standard organic dyes (FITC, Alexa Fluor dyes). Do not use with fluorescent proteins or phalloidin. | |||
Readout | Fluorescent signal accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period | |||
Format | 10 plates | 10 plates | 10 plates | 10 plates |
Cat. No. | C10351 | C10353 | C10355 | C10357 |
Click-iT EdU Colorimetric IHC Detection Kit | |
---|---|
Label | Biotin |
Bibliography | Citations |
Protocol | Microscopy |
Sample type | Optimized for tissue sections |
Multiplexing | Commonly used secondary tissue stains such as hematoxylin or methyl green can be used to review the cellular context surrounding the proliferation signal. |
Readout | The peroxidase enzyme and subsequent colorimetric precipitation accumulates in the nuclei of cells where DNA has been synthesized during the EdU incubation period. The color will be brown. |
Format | 1 kit/50 slides |
Cat. No. | C10644 |
Figure 1. BrdU, Click-iT EdU, and Click-iT Plus EdU staining. A375 melanoma cells expressing GFP were imaged for cell proliferation using (A) traditional antibody-based BrdU, (B) Click-iT EdU, and (C) Click-iT Plus EdU. The nucleus is stained blue with Hoechst dye; pink indicates the proliferation signal; green indicates GFP.
Click-iT labeling employs bioorthogonal reactive chemistry with reaction partners, including EdU, that are not endogenously present in biological molecules, cells, tissues, or model organisms. Click chemistry can be used to study cell proliferation through modification of a nucleoside analog that is incorporated during DNA synthesis of actively dividing cells.
Click-iT EdU and Click-iT Plus EdU assays are an alternative to BrdU (5-bromo-2’-deoxyuridine) staining for cell proliferation using a versatile, bioorthogonal, and specific labeling reaction. The Click-iT technology advantage is in the chemistry—small and unique with low-background labeling. The azide-alkyne click reaction is a “spring loaded” reaction that happens quickly and forms a single product with high yield and a strong covalent bond. Given the mild reaction conditions of Click-iT assays, these kits can assess cell proliferation while preserving cell morphology, DNA integrity, antigen binding sites, and fluorescent signal for better DNA staining to examine cell cycles.
In the traditional BrdU assay, BrdU is incorporated into the newly synthesized DNA of proliferating cells which are identified using an anti-BrdU antibody. DNA denaturation using HCl, heat, or digestion with DNase is required to expose the incorporated BrdU to the anti-BrdU antibody for detection. This process can lead to unwanted artifacts due to the harsh conditions and extend the time to detection. Such severe treatments can result in inconsistent staining and decreased signal. Unlike BrdU, EdU can be easily detected after DNA incorporation due to the small size of the fluorescent azide and does not require an antibody or DNA denaturation for detection. Thus, use of Click-iT EdU cell proliferation assays preserve 3-D protein structure and function with reduced target interference better than traditional BrdU staining. With their mild reaction conditions, Click-iT EdU cell proliferation kits allow for more consistent results, faster protocols, and better signal to noise compared to an antibody-based BrdU cell proliferation assay.
The simplicity of the click detection method makes the EdU assay a faster alternative to the BrdU assay.
5-bromo-2’- deoxyuridine (BrdU) is a synthetic analog of thymidine which incorporates into newly synthesized DNA. Proliferating cells labeled with BrdU can be identified using an antibody-based BrdU staining protocol with a flow cytometer or BrdU labeling and detection with a microscope. BrdU staining can be combined with cell surface and/or intracellular targets to help understand cellular function. BrdU and EdU can be used in the same experiment. To minimize cross-reactivity between BrdU and EdU staining, we recommend anti-BrdU clone MoBU-1.
Figure 2. BrdU staining with antibody detection. Denaturation is required for the 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, the benefits of the Click-iT EdU assay and Click-iT Plus EdU assay for cell proliferation are clear.
Figure 3. 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.
The Click-iT EdU Cell Proliferation Kits utilize copper-mediated azide-alkyne click cycloaddition for EdU detection. Given that copper can damage proteins and nucleic acids, Click-iT Plus EdU Cell Proliferation Kits were developed and include Invitrogen Alexa Fluor picolyl azides for EdU detection in the presence of copper-sensitive compounds. Picolyl azides react efficiently with chelated copper, so free copper in the click reaction is minimized, protecting against undesired interactions with proteins (e.g., green fluorescent protein (GFP), R-phycoerythrin (R-PE)), nucleic acids (e.g., RNA, oligos), and small molecules. Picolyl azides offer increased sensitivity and faster reaction times than standard azides, and allow multiplexing while retaining the benefits of the standard azide-alkyne click reaction (Table 3). Click-iT Plus assays can determine cell proliferation while preserving cell morphology, DNA integrity, antigen-binding sites, and fluorescent signals for better DNA staining to examine cell cycles.
Classic Click-iT EdU | Click-iT Plus EdU | |
---|---|---|
Detection label functional group | Azide | Picolyl azide |
Signal intensity | Bright | As bright or brighter |
Reaction rate | Fast | As fast as azide or faster |
Organic dyes such as Alexa Fluor dyes and fluorescein (FITC) | ✔ | ✔ |
Fluorescent protein including GFP and mCherry compatibility | Not recommended | ✔ |
Phalloidin compatibility | Not recommended | Not recommended |
Qdot compatibility | Post click staining | Post click staining |
R-PE and R-PE tandem compatibility | Post click staining | ✔ |
PerCP, allophycocyanin (APC) and APC-based tandems (i.e., Alexa Fluor 680-APC) | ✔ | ✔ |
Polymer dyes including Brilliant Violet dyes compatibility | ✔ | ✔ |
Cell proliferation is traditionally assessed by incubating cells with a single “pulse” of a nucleoside analog that is incorporated into the DNA of actively dividing cells, followed by detection using radioactivity, antibodies, or click chemistry. Many applications benefit from the incorporation of two different analogs at different time points (dual-pulse labeling), which can further distinguish cell proliferation and define cell cycle kinetics. Dual-pulse labeling for cell proliferation is commonly done with BrdU and another analog such as 5-chloro-2´-deoxyuridine (CldU) or 5-iodo-2´- deoxyuridine (IdU), which also require antibodies that have high antigen specificity without cross-reactivity.
The BrdU labeling technique can be combined with click chemistry detection for a simplified method of dual-pulse labeling using EdU when utilizing highly specific anti-BrdU antibodies. For dual-pulse applications, the use of EdU as one of the nucleoside analogs greatly simplifies the procedure, as there is no reactivity between the Click-iT azide detection reagent and the incorporated BrdU. Moreover, several anti-BrdU antibodies, including the MoBU-1 clone, do not cross-react with EdU.
Note: BrdU is preferentially incorporated over EdU, therefore requiring EdU to be administered prior to BrdU in dual-pulse labeling.
Figure 4. Example dual pulse BrdU and EdU staining. Immunofluorescence for EdU (green) and BrdU (red). Data generated by: Gómez-Nicola D, Valle-Argos B, Pallas-Bazarra N, Nieto-Sampedro M. Interleukin-15 regulates proliferation and self-renewal of adult neural stem cells. Mol Biol Cell. 2011 Jun 15;22(12):1960-70. PMID: 21508317
Figure 5. 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 (C10632), followed by staining with Invitrogen FxCycle Violet Stain (F10347). 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 6. Cell proliferation multicolor imaging with Click-iT EdU. Muntjac cells pulsed with EdU. Cells were subsequently fixed and permeabilized and labeled DNA detected using Click-iT EdU Alexa Fluor 647 kit (C10085). Tubulin (in blue) is labeled with a mouse primary followed by Alexa Fluor 350 goat anti–mouse IgG antibody. Golgi (in green) is labeled with Alexa Fluor 488 conjugate of lectin HPA (L11271), and peroxisomes (in orange) are labeled with a rabbit primary followed by Alexa Fluor 555 donkey anti–rabbit IgG antibody.
Figure 7. Click-iT EdU enables detection of DNA synthesis on plant cells without cell wall digestion, currently not possible with BrdU. Detection of DNA synthesis on undigested Medicago sativa (alfalfa) suspension cultures with Click-iT EdU (green). Nuclei counterstained with DAPI (blue). Colocalizing regions appear as cyan. Laser scanning confocal microscopy was performed on Olympus Fluoview FV1000 confocal microscope equipped with 488 nm argon laser (used for Alexa Fluor 488 dye detection) and near-UV 405 nm laser (for DAPI detection). UPLSAPO 60X Oil immersion objective (NA:1.35) was used for image acquisition. Six confocal sections of 1.28 µm/slice were merged onto differential interference contrast image.
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