Invitrogen™ Click-iT™ TUNEL Colorimetric IHC Detection Kit

Catalog No.	Quantity
C10625	1 Kit

Catalog No. C10625 Supplier Invitrogen™ Supplier No. C10625

Description

Click-iT TUNEL Colorimetric IHC Detection Kit

The Click-iT™ TUNEL Colorimetric IHC Detection Kit is used to identify apoptotic cells in tissue and cultured cell samples through the use of a small, highly specific labeling moiety and streptavidin-peroxidase conjugation. After incorporation of the labeling moiety into sites of DNA fragmentation, detection is achieved through a catalyzed 'click' reaction that adds a biotin group at these sites. The subsequent addition of a streptavidin-peroxidase and peroxidase substrate results in a dark brown signal that can be detected with light microscopy and stored for future analysis.

Find more tools for image-based detection of apoptotic cells >

Optimized for the detection of apoptotic cells in either tissue or cell samples

Improved colorimetric TUNEL assay—better label incorporation due to small reactive moiety

Increased sensitivity—specific label incorporation results in lower background and brighter signal

Content-rich results—cell morphology, cellular environment, and apoptotic signal are clearly visible

Flexibility—the assay can be configured for 50 independent TUNEL apoptosis tests

The later stages of apoptosis are characterized by changes in nuclear morphology, chromatin condensation, nuclear envelope degradation, and DNA fragmentation. Terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assays are based on the incorporation of modified dUTPs by terminal deoxynucleotidyl transferase (TdT) at the 3’-OH ends of fragmented DNA. Colorimetric TUNEL assays utilize dUTPs conjugated with a biotin moiety, followed by the addition of a streptavidin-peroxidase conjugate and peroxidase substrate, resulting in a dark brown apoptotic signal. However, colorimetric TUNEL assays are prone to high background, which reduces the sensitivity and specificity of the signal.

The Click-iT TUNEL Colorimetric IHC Detection Kit was developed to address these issues by utilizing a dUTP modified with an alkyne group (a small bio-orthogonal functional group) that enables the nucleotide to be more readily incorporated by TdT. After incorporation, a highly specific click reaction covalently links biotin azide and the alkyne group. Streptavidin-peroxidase (horseradish peroxidase) followed by peroxidase substrate (DAB) are added, allowing colorimetric detection of apoptotic cells. The high degree of labeling specificity inherent in the click technology results in low background and improved detection of apoptotic cells.

The Click-iT TUNEL Colorimetric IHC Detection Kit has been optimized and contains all of the reagents needed for detection of apoptotic cells from either tissue or cell samples. The reagents supplied in this kit can be used to test 50 samples and configured to test from one to 50 samples at a time.

Specifications

• Quantity: 1 Kit
• Format: Slide
• Product Type: TUNEL Colorimetric IHC Detection Kit
• Content And Storage: Mixed storage conditions for kit components: 2–8°C and -5 to -30°C
• Shipping Condition: Dry Ice
• Product Line: Click-iT
• Detection Method: Colorimetric
• For Use With (Equipment): EVOS™ XL Core Imaging System, EVOS™ XL Imaging System

Frequently Asked Questions (FAQs)

Can Metal Enhanced DAB Substrate Kit (Cat No. 34065) be used instead of the DAB provided in the Click-iT TUNEL Colorimetric IHC Detection Kit?

Yes, but we have not optimized its use with this kit.

Can the Click-iT TUNEL Colorimetric IHC Detection Kit be used with other colorimetric dyes?

Yes, other colorimetric stains can be used.

I ran out of the TdT enzyme used in the Click-iT TUNEL assay. Can I purchase it separately?

Yes, additional Terminal Deoxynucleotidyl Transferase (rTdT) can be purchased as Cat. No. 10533-065 or 10533-075. Additional TdT reaction buffer can be purchased as Cat. No. 16314-015.

Can I perform Click-iT EdU TUNEL detection on cells growing in 3D culture?

We have not validated the use of EdU TUNEL for apoptosis detection in 3D culture systems, but as this reagent is compatible for labeling cells in vivo, it is also expected to label cells in 3D culture systems. There are a number of reports in the literature that use this product in 3D culture systems; here are some citations:

Lei Y, Schaffer DV (2013) A fully defined and scalable 3D culture system for human pluripotent stem cell expansion and differentiation. Proc Natl Acad Sci U S A 110:E5039-E5048.
Derda R, Laromaine A, Mammoto A et al. (2009) Paper-supported 3D cell culture for tissue-based bioassays. Proc Natl Acad Sci U S A 106:18457-18462.
Robertson FM, Ogasawara MA, Ye Z et al. (2010) Imaging and Analysis of 3D Tumor Spheroids Enriched for a Cancer Stem Cell Phenotype. J Biomol Screen 15:820-829.

For the Click-iT TUNEL for In Situ Apoptosis Detection Assay, how long should I digest my tissue samples in proteinase K?

Most tissue samples will be adequately digested in 15 minutes. The optimal incubation time will vary depending on tissue type and thickness. We have observed that brain tissue needs longer proteinase K treatment than other tissues tested.

How thick can my tissue sections be for apoptosis detection using the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay?

We have validated the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay on 5-20 µM thick FFPE sections of mouse intestine, kidney, liver, heart, and colon.

Can I use the original Click-iT TUNEL assay kits on tissue samples?

The original Click-iT TUNEL assay kits were optimized for cell culture samples and may also be used on tissue samples. We recommend using the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay kits on tissue samples; the protocols for the Click-iT Plus TUNEL for In Situ Apoptosis Detection assay kits have been optimized for tissue. The protocol was modified to improve accessibility of the TdT enzyme into the multiple cell layers of tissue samples. In addition, we found that the original Click-iT TUNEL kit may show higher non-specific binding and punctate staining in tissues compared to the Click-iT Plus TUNEL kit. One method that works well to increase permeability for tissues is to replace the detergent permeabilization step with proteinase K digestion and then refix the sample in formaldehye. The Tissue Fixation and Permeabilization protocol in section 3 of the Click-iT Plus TUNEL kit manual can be followed for tissue samples using the original Click-iT TUNEL assay. Pepsin and other proteolytic enzymes can also be used to improve permeability.

What are the advantages of flow cytometry?

-Measures data from single cells.
-Data are obtained for a large number of cells, generating a rich statistical analysis of cell populations.
-Because single cells are measured, it will reveal heterogeneity within a population.
-With the ability to multiplex, small sub-populations can be identified.
-Thousands of cells can be analyzed rapidly.
-It is ideally suited for blood samples and other cells in suspension.
-Data can be re-analyzed multiple times after acquisition.
-Flow cytometry files (FCS) can be archived.

What kinds of applications can I run on a flow cytometer?

There are several applications, some of which include immunophenotyping, cell cycle analysis, apoptosis assays such as annexin V staining, CellEvent Caspase-3/7 assay, and TUNEL assay, cell viability, proliferation assays such as CellTrace assay and Click-iT EdU assay, measurements of mitochondrial potential with MitoProbe assays, and cell counting using counting beads.

For Research Use Only. Not for use in diagnostic procedures.