Invitrogen™ Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit
On demand
Order number :
C10418
Invitrogen™ Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit
The Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells as compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the 405 nm laser of the flow cytometer.
Features
- 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, i.e., 3H-thymidine. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT™ EdU Flow Cytometry Assay Kits are novel alternatives to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog which is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry: 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 Alexa Fluor ™ 488, Alexa Fluor™ 647, or Pacific Blue™ dyes. 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 advantages of Click-iT™ EdU labeling are readily evident while performing the assay. 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™ 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 may 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 EdU cell proliferation kit is compatible with cell cycle dyes. This EdU assay kit can also be multiplexed with antibodies against surface and intracellular markers.
- Quick and Simple Protocol, The
Click-iT™ EdU protocol is based on the aldehyde fixation and detergent
permeabilization steps for immunohistochemical antibody labeling, but
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:
• Treat cells with EdUM.
• Fix and permeabilize cells.
• Detect S-phase cells with Click-iT™ detection cocktail for 30 min.
• Wash once.
• Analyze. - Get Accurate Results Economically, By increasing the number of assays per kit, the Click-IT™ EdU Pacific Blue™ Flow Cytometry Assay Kit is less expensive than the traditional BrdU assays making them ideal for large experiments.
Contents
- EdU (5-ethynyl-2' -deoxyuridine).
- Pacific Blue™ azide.
- anhydrous dimethylsulfoxide (DMSO).
- Click-iT™ fixative.
- Click-iT™ saponin-based permeabilization and wash buffer.
- copper (II) sulfate.
- Click-iT™ EdU buffer additive.
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