EdU assay protocol.pdf - Click-iTu00ae EdU Imaging Kits...

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MAN0002026 Revised: 29–July–2011 |MP 10338Click-iT® EdU Imaging Kits Table 1. Contents and storage information.MaterialC10337C10338C10339C10340ConcentrationStorage*EdU (Component A)5 mg5 mg5 mg5 mgNA• 2–6˚C• Desiccate• Protect from light• DO NOT FREEZEAlexa Fluor® azide (Component B)1 vial (Alexa Fluor® 488)1 vial (Alexa Fluor® 555)1 vial (Alexa Fluor® 594)1 vial (Alexa Fluor® 647)NADimethylsulfoxide (DMSO, Component C)4 mL4 mL4 mL4 mLNAClick-iT® EdU reaction buffer (Component D)4 mL4 mL4 mL4 mL10X solution containing Tris-buffered salineCuSO4(Component E)1 vial1 vial1 vial1 vial100 mM aqueous solutionClick-iT® EdU buffer additive (Component F)400 mg400 mg400 mg400 mgNAHoechst 33342 (Component G)35 µL35 µL35 µL35 µL10 mg/mL in water*These storage conditions are appropriate when storing the entire kit upon receipt. For optimal storage conditions for each component, see labels on the vials. When stored as directed, this kit is stable for 1 year. NA = Not applicable.Number of assays:Sufficient material is supplied for 50 coverslips based on the protocol below. Approximate fluorescence excitation/emission maxima, in nm: Alexa Fluor® 488 azide:495/519; Alexa Fluor® 555 azide:555/565; Alexa Fluor® 594 azide: 590/615; Alexa Fluor® 647 azide: 650/670; Hoechst 33342: 350/461, bound to DNA.IntroductionMeasuring a cell’s ability to proliferate is a fundamental method for assessing cell health, determining genotoxicity, and evaluating anti-cancer drugs. The most accurate method of doing this is by directly measuring DNA synthesis. Initially this was performed by incorporation of radioactive nucleosides3 (for example, H-thymidine). This method was replaced by antibody-based detection of the nucleoside analog bromo-deoxyuridine (BrdU). The Click-iT® EdU Assay from Invitrogen is a novel alternative to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) provided in the kit is a nucleoside analog of thymidine and is incorporated into DNA during active DNA synthesis.1Detection is based on a click reaction,2-5a copper-catalyzed covalent reaction between an azide and an alkyne. In this Search the handbookBuy Now
Click-iT® EdU Imaging Kits |2application, the EdU contains the alkyne and the Alexa Fluor® dye contains the azide. The advantages of the 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. 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 (typically using HCl or heat or digestion with DNase) to expose the BrdU so that it may be detected with an anti-BrdU antibody (Figure 1).The denaturation step in the BrdU protocol can disrupt dsDNA integrity, which can affect nuclear counterstaining, and can also destroy cell morphology and antigen recognition sites. In contrast, the EdU assay kit is not only easy to use, but is fully compatible with DNA

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