The Click-iT® EdU Microplate Assay is a superior alternative to traditional cell proliferation assays that is optimized for microplate-based fluorescence analysis. This assay provides a simple and rapid workflow with reduced wash steps compared to traditional BrdU colorimetric or fluorescent proliferation assays. In this assay the modified thymidine analogue EdU is efficiently incorporated into newly synthesized DNA and fluorescently labeled with bright, photostable Amplex® UltraRed dye in a fast, highly-specific click reaction. This fluorescent labeling of proliferating cells is accurate and compatible with antibody methods due to the mild click protocol.
• Simple—works the first time, every time, in less time
• Efficient—no denaturation steps or harsh treatment required
• Sensitive—outstanding sensitivity and dynamic range
• Versatile—fluorescence or absorbance readouts through use of Amplex® UltraRed reagent
The most accurate proliferation-detection methods are based on the incorporation and measurement of nucleoside analogues in newly synthesized DNA, with bromodeoxyuridine (BrdU) a commonly used analogue. BrdU-labeled DNA is quantitated using anti-BrdU antibodies following DNA denaturation by harsh methods (HCl, heat, or enzymes) to expose the BrdU molecules. This step is time consuming and difficult to perform consistently.Superior Proliferation Methodology
The Click-iT® EdU Microplate Assay provides a superior alternative to BrdU assays for measuring cell proliferation. EdU (5-ethynyl-2'-deoxyuridine) is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis. With Click-iT® EdU, mild fixation and detergent permeabilization is sufficient for the small molecule-based Click-iT® EdU detection reagent to gain access to the DNA. The signal amplification steps include incubation with an anti-Oregon Green® antibody conjugated to horseradish peroxidase (HRP), which then reacts with Amplex® UltraRed substrate in a 1:1 stoichiometry and produces a bright red, fluorescent product (excitation/emission maxima ~568/585 nm).
With a high extinction coefficient, good quantum efficiency, and resistance to auto-oxidation, Amplex® UltraRed reagent delivers higher sensitivity and a broader assay range than other fluorogenic or colorimetric peroxidase substrates. Amplex® UltraRed reagent also offers versatility through detection by either fluorescence or absorbance measurement.
This kit also includes Amplex® UltraRed stop reagent that enables the peroxidase reaction to be terminated at a user-determined time point and stabilizes the signal for up to 24 hours. As a consequence, the Click-iT® EdU Microplate Assay is not only easy to use, but more accurate and compatible with cell cycle analysis and other intracellular or extracellular targets for truly content-rich results.
The Click-iT® EdU Microplate Assay is optimized for microplate-based fluorescence analysis; visit the Click-iT® technology area of our website for kits designed for fluorescence microscopy, high-content imaging, or flow cytometry platforms.Learn more about Click-iT® technologyNotes:
The Click-iT® assay can be used on cells in culture or in vivo
following administration of EdU by feeding or injection methods.
The Click-iT® assay can be used with BrdU in dual pulse experiments by using the anti-BrdU (clone MoBu-1) antibody, which does not cross react with EdU.
The Click-iT® technology is compatible with immunohistochemical, immunocytochemical, and fluorescent dyes that are fixation tolerant or designed for fixed-cell labeling.