Invitrogen™ Click-iT™ EdU Proliferation Assay for Microplates

Description

• After incorporation of EdU (a modified thymidine analogue) into newly synthesize DNA, horse radish peroxidase (HRP) is covalently attached via the highly specific click reaction
• Amplex UltraRed Reagent, an HRP substrate, is then added and the resulting fluorescence signal detected
• Because of the highly specific attachment of HRP to EdU, this assay results in highly sensitive and reliable measurement of mammalian cell proliferation in a microplate format
• Click-iT EdU Proliferation Assay for Microplates features include: • Improved performance —direct conjugation of HRP to incorporated EdU improves assay sensitivity and accuracy • Increased reliability —assay format results in low well-to-well variability • Simplified protocol —results in reduced time to data; easier to follow protocol • Multiplex enabled —assay can be easily multiplexed with other cell health reagents resulting in more data per sample Measuring cell proliferation is a fundamental method of assessing cell health, determining genotoxicity, and evaluating anti-cancer drugs
• The most accurate method of measuring proliferation is by direct detection of DNA synthesis
• Originally this was accomplished through incorporation of a radioactive nucleoside (e.g., 3 H-thymidine)
• This method was replaced by antibody-based detection of the nucleoside analog bromo-deoxyuridine (BrdU)
• After incorporation of BrdU into the newly synthesized DNA, denaturation is required to expose the BrdU molecules to the anti-BrdU antibody
• The denaturation involves harsh methods (HCl, heat, or enzymes) and is time consuming and difficult to perform consistently
• The Click-iT EdU Proliferation Assay is an alternative to the BrdU assay
• The nucleoside analog EdU (5-ethynyl-2´-deoxyuridine) is added to live cells and incorporated into DNA during active DNA synthesis
• The incorporated EdU contains an alkyne group which is covalently joined to the azide group present in HRP using a highly specific copper-catalyzed covalent reaction ('click reaction')
• Amplex UltraRed Reagent is then added and its conversion by HRP into a highly fluorescent product is recorded using a fluorescence microplate reader (excitation: 568, emission: 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 HRP substrates
• It also offers versatility through detection by either fluorescence or absorbance measurement.