The Click-iT® TUNEL Alexa Fluor® 488 Imaging Assay utilizes a dUTP modified with an alkyne, a small, bio-orthogonal functional group that enables the nucleotide to be more readily incorporated by TdT than other modified nucleotides, including BrdUTP, biotin-dUTP or fluorescein-dUTP. Detection is based on a click reaction, a copper (I) catalyzed reaction between an azide and alkyne. Click chemistry fills the void when methods such as direct labeling or the use of antibodies are not efficient. The small size of the detection reagent, an Alexa Fluor® azide (MW <~1000) compared to that of an antibody (MW ~150,000) enables facile penetration of complex samples with only mild fixation and permeabilization required. Compared to other assays using one of the other modified nucleotides, the Click-iT® TUNEL assay is able to detect a higher percentage of apoptotic cells under identical conditions. The assay is also fast and is complete within 2 hours. Furthermore, the Click-iT® TUNEL assay allows multiplexing with surface and intracellular biomarker detection.
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