The Click-iT Plus TUNEL Alexa Fluor 488, 594, and 647 assays for in situ apoptosis detection can detect apoptotic cells in tissue and cultured cell samples through the incorporation of a small, highly specific labeling moiety and a bright fluorescent dye. After incorporation of the labeling moiety into DNA fragments, detection is achieved through a catalyzed “click” reaction using conditions mild enough to preserve the emitted fluorescent signal from GFP or RFP.

Other advantages of the Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection include:
• Optimized for the detection of apoptotic cells in either tissue or cell samples
• Multiplex enabled—optimized to work with fluorescent dyes or proteins such as GFP and RFP
• Improved TUNEL assay—better label incorporation due to small reactive moiety
• Bright apoptotic signal—uses Alexa Fluor dyes, resulting in a stable, non-photobleaching fluorescent signal
• Flexibility—the assay can be configured to test 1–50 samples at a time

Fragmentation of cellular DNA is a hallmark of apoptosis. The TUNEL assay is the most widely used method to detect fragmented DNA in apoptotic cells or tissue samples. The TUNEL assay begins with incorporation of modified dUTP at the 3’-OH end of the fragmented DNA. The dUTP modification is often the addition of a fluorophore. Due to the size of the fluorophore, the modified dUTP can display lower than expected incorporation rates, which can affect the sensitivity of the TUNEL assay. Additionally, many fluorophores used in currently available TUNEL assay kits suffer from photobleaching and fluorescent spectral overlap issues, both of which reduce the sensitivity of and ability to multiplex the assay.

The Click-iT Plus TUNEL assay was developed to address these issues. The assay uses dUTP modified with an alkyne group (a small bio-orthogonal functional group), allowing the nucleotide to be more readily incorporated. After incorporation, a highly specific click reaction between the alkyne group and an Alexa Fluor picolyl azide fluorescent dye, and subsequent detection of that dye, results in a sensitive and specific assay for the detection of apoptotic cells or tissue samples. Because of its gentle reaction conditions, the Click-iT Plus TUNEL assay enables multiplexing with fluorescent proteins or dyes.

The Click-iT Plus TUNEL assay has been validated with several different formalin-fixed paraffin-embedded tissue types. In all cases, its ability to multiplex with fluorescent proteins and dyes was preserved. Additionally, the ability to stain actin using fluorescent-labeled phalloidin was also preserved.

The Click-iT Plus TUNEL assay contains all the reagents needed to detect apoptotic cells from either tissue or cell samples. The reagents supplied in this kit can be used to test 50 samples and can be configured to test 1–50 samples at a time.