Click-iT™ EdU Proliferation Assay for Microplates - FAQs

If the Amplex UltraRed reagent was spontaneously oxidized, all samples would have high levels of signal. The lack of any signal indicates that some component in the reaction is non-functional or little or no EdU was incorporated by the cells. The HRP-azide (Component B), the Click-iT EdU reaction additive (Component F), and the hydrogen peroxide (Component J) are the most labile components in the kit. HRP can be denatured and inhibited by sodium azide. The Click-iT Edu reaction additive (Component F) should be diluted only shortly before use and dilute solutions should not be stored. Hydrogen peroxide does spontaneously degrade over time, but more readily when diluted; prepare the hydrogen peroxide stock solution shortly before use and do not store.

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This may be possible, but the final working concentrations and incubation steps would have to be optimized by the user. Be advised that chromogenic substrates are not as sensitive as the fluorogenic Amplex UltraRed reagent.

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The Click-iT EdU Cell Proliferation Assay for Microplates protocol requires samples to be fixed and permeabilized prior to the click reaction. Most endogenous peroxidases may be inactivated after fixation/permeabilization and after the click reaction. If this is of concern, apply the Amplex UltraRed reaction mixture without added H2O2 to the samples after fixation, but before the click reaction (i.e., no HRP-azide added to the sample). In the absence of added H2O2 and HRP-azide, if you see any change in Amplex UltraRed fluorescence intensity, this is indicative of endogenous peroxidase activity.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.