Apoptosis is marked by defined cellular processes, including cell rounding, blebbing, and DNA fragmentation. The TUNEL assay is the most widely used analytical method to detect fragmented DNA in apoptotic cells in tissue samples, beginning with the incorporation of modified dUTPs at the 3’-OH ends of fragmented DNA. The dUTPs often include a fluorophore molecule. Due to the size of the fluorophore, modified dUTPs can display lower than expected incorporation rates, negatively affecting the sensitivity of the TUNEL assay. Quite often, fluorophores used in currently available TUNEL assay kits exhibit photobleaching and fluorescent spectral overlap issues, both of which reduce the sensitivity of and ability to multiplex these assays.

The Click-iT Plus TUNEL assay was developed to address these issues and is based on a copper (I) catalyzed (i.e., click) reaction between an azide and alkyne. The small size of the Alexa Fluor azide, which has a molecular weight of about 1 kDa, enables easier incorporation of the Alexa Fluor 488, 594, or 647 dye into complex samples, as compared to larger (MW ˜100,000 Da) antibodies. This permits mild fixation or permeabilization and faster assay turnaround time (about two hours) and enables the detection of a higher percentage of apoptotic cells under identical conditions. Because of its gentle reaction conditions, the Click-iT Plus TUNEL assay can also be multiplexed with fluorescent proteins or dyes.

The Click-iT TUNEL Alexa Fluor imaging assays contain all components needed to accurately and reliably detect apoptosis in adherent cells grown on coverslips or 96-well microplates, and they also include DNase I to generate strand breaks for the positive control.