Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit, 50 rxn(s)
Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit, 50 rxn(s)
Invitrogen™

Click-iT™ Plus EdU Alexa Fluor™ 647 Flow Cytometry Assay Kit, 50 rxn(s)

Green features
The Click-iT® Plus EdU Alexa Fluor® 647 Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNARead more
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Catalog NumberQuantity
C1063450 assays
Catalog number C10634
Price (USD)
772.00
Each
-
Add to cart
Quantity:
50 assays
Price (USD)
772.00
Each
Add to cart
The Click-iT® Plus EdU Alexa Fluor® 647 Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the 647 nm laser of the flow cytometer. The Click-iT® Plus formulation is compatible with standard fluorophores, including R-PE and R-PE tandems, as well as fluorescent proteins.

• Multiplexable—compatible with R-PE (and tandems) and fluorescent proteins
• Accurate—superior results compared to BrdU assays
• Fast—results in as little as 60 minutes

View selection guide for all Click-iT™ EdU and Click-iT™ Plus EdU assays for flow cytometry.

Multiplexable
The Click-iT® Plus formulation provides increased multiplexibility 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.

An Advanced Method Giving You Results Superior to BrdU
The most accurate method of proliferation analysis is direct measurement of DNA synthesis. Originally, this was performed through incorporation of radioactive nucleosides. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT® Plus EdU Flow Cytometry assay is a novel alternative to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog that is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry, which is a copper-catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to the Alexa Fluor® dye. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population.

Mild Conditions Allow Use with Cell Cycle Dyes and Antibodies
The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT® Plus detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it can be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution, as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use Click-iT® Plus EdU assay is compatible with cell cycle dyes. The Click-iT® Plus EdU assay can also be multiplexed with antibodies against surface and intracellular markers, as well as conjugates labeled with standard fluorophores including R-PE, R-PE tandems, and fluorescent proteins (GFP and mCherry).

Quick and Simple Protocol
The Click-iT® Plus EdU protocol is based on the aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling. However, EdU is compatible with other fixation/permeabilization agents including saponin and methanol. 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® Plus detection cocktail for 30 min.
4. Wash once.
5. Analyze.

Results can be seen in as little as 60 minutes in some circumstances, but we recommend 90 minutes for all applications.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
Detection MethodFluorescence
Dye TypeAlexa Fluor™ 647
FormatTube(s)
Green FeaturesLess hazardous
Quantity50 assays
Shipping ConditionRoom Temperature
Emission650/670
For Use With (Equipment)Flow Cytometer
Product LineClick-iT
Product TypeFlow Cytometry Assay Kit
Unit SizeEach
Contents & Storage
Contains EdU (5-ethynyl-2' -deoxyuridine), Alexa Fluor™ 647 picolyl azide, anhydrous dimethylsulfoxide (DMSO), Click-iT™ fixative, Click-iT™ saponin-based permeabilization and wash buffer, Copper Protectant, and Click-iT™ EdU buffer additive.
  • Store at 2°C to 8°C
  • Dessicate and protect from light.
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    Frequently asked questions (FAQs)

    What is the fluorescence excitation and emission maxima of Alexa Fluor 647 dye?

    Alexa Fluor 647 has an excitation/emission maxima of 650/670 nm.

    Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

    What are the main characteristics of a Click-iT reaction?

    Click reactions have several general characteristics: the reaction is efficient, no extreme temperatures or solvents are required, the reaction is complete within 30 minutes, the components of the reaction are bio-inert, and perhaps most importantly, no side reactions occur-the label and detection tags react selectively and specifically with one another. This final point is a key advantage of this powerful detection technique; it is possible to apply click chemistry-labeled molecules to complex biological samples and detect them with unprecedented sensitivity due to the extremely low background of the reaction.

    Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

    I will be performing a cell proliferation assay using Click-iT EdU kit. At what point can I stop overnight, or do I have to perform all the steps continuously?

    One may store the sample after fixation overnight in PBS at 4oC. For longer storage (<1 week) , store in buffer with 1-2% formaldehyde or in formalin to limit microbial growth. If you use sodium azide as a microbial inhibitor, it must be completely removed prior to the Click-iT reaction.

    Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center.

    Can I combine Click-iT or Click-iT Plus reactions with phalloidin conjugates used for actin staining?

    We do not recommend using phalloidin conjugates for staining actin in combination with traditional Click-iT or Click-iT Plus reactions since phalloidin is extremely sensitive to the presence of copper.

    For staining actin in combination with traditional Click-iT or Click-iT Plus reactions, we recommend using anti-α-actin antibodies for staining actin in the cytoskeleton. You can find a list of our actin antibodies here.

    Another option would be to use the Click-iT Plus Alexa Fluor Picolyl Azide Toolkit (Cat. Nos. C10641, C10642, C10643). These Click-iT Plus toolkits provide Copper and Copper protectant separately which makes it easier to titrate the copper concentration to obtain optimal labeling with minimal copper-mediated damage. You may need to optimize the click reaction with the lowest possible concentration of copper and then perform the phalloidin staining.

    Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

    With the Click-iT Plus EdU flow cytometry kits, my live-cell sample includes EDTA in the buffer/medium. Would this cause any problems with EdU incorporation or the click reaction?

    The presence of EDTA in the cell buffer/media would not be an issue for EdU incorporation and it should be mostly gone from the sample after fixation and permeabilization. EDTA must not be present during the click reaction.

    Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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