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View additional product information for Click-iT™ EdU Microplate Assay - FAQs (C10214)
5 product FAQs found
Yes, EdU is frequently used for in vivo assays and acceptable EdU incorporation can be obtained following injection or media incubation. Optimal EdU incubation times and concentrations will depend on the incorporation method and the organism/tissue type. Recommendations on EdU incorporation methods in various model organisms and protocols for detection of EdU in tissue samples can be found in the In Vivo Use of Click-iT EdU Cell Proliferation Assays application note and the Click-iT EdU Labeling In Vivo Cell Proliferation Protocol.
Table 2 of the EdU (5-ethynyl-2'-deoxyuridine) product manual also provides some recommendations. Methods that have been optimized for BrdU labeling for your organism are a good starting point. Tissue samples can be frozen or paraffin-embedded and then processed using normal procedures for subsequent IHC staining. For detection of EdU, use any desired method for fixation and permeabilization, and then perform the click detection reaction exactly according to the protocol for cultured cells in the product manual.
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The Click-iT EdU flow kits use a lower azide dye concentration than what is optimal for the EdU imaging kits, so it is not possible to use a flow kit to label cells for an imaging experiment. It is possible to use a Click-iT EdU imaging kit for samples for flow analysis, but it would be necessary to optimize the azide dye concentration first. The azide dye concentrations used in the Click-iT EdU kits are proprietary.
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The copper sulfate solution component supplied in the Click-iT kits is 100 mM copper sulfate in water. Copper (II) sulfate (CuSO4) powder is available from many chemical suppliers.
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Unfortunately, the actual concentration of the azide dyes used in the Click-iT assay kits is proprietary, so we cannot provide the exact concentration used. For a general guideline, we recommend making up a 1-10 mM DMSO stock solution and using at approximately 1-10 µM final concentration in the click reaction. Too high of a dye concentration can result in high nonspecific background or dye aggregates; too low of a concentration may not give a strong enough signal.
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If you are using EdU or EU, the simplest thing to do is purchase a complete kit that is specific for the type of assay you are performing, either flow, imaging, or a microplate assay. The complete kits contain the azide detection reagent as well as copper and the buffers necessary to perform the click reaction. If you have a different alkyne metabolite analog or do not wish to purchase EdU or EU, then you will need to purchase any of our azide dye detection reagents and the Click-iT Cell Reaction Buffer Kit (Cat. No. C10269), which contains the copper solution and the other buffer reagents necessary to perform the click reaction for flow and imaging assays. If you wish to perform a microplate assay, then you are limited to the Click-iT EdU Microplate Assay (Cat. No. C10214), which provides all the reagents necessary for the click reaction as well as the reagents necessary to perform the amplification of the signal so that it can be detected on a microplate reader.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.