Invitrogen™ Click-iT™ Plus TUNEL Assay Kits for In Situ Apoptosis Detection

Product Code.	Color	Label or Dye	unitSize
15471094	Far-Red	Alexa Fluor™ 647	1 set
15350244	Green	Alexa Fluor™ 488	1 set
15313038	Red	Alexa Fluor™ 594	Each

Product Code. 15471094 Supplier Invitrogen™ Supplier No. C10619

Description

Detect apoptosis in cells and tissues samples with Click-iT Plus TUNEL Assay kits, which offer easy dye incorporation and can be multiplexed with GFP and RFP.

Detect more apoptotic cells in tissues and cultured cell samples with the Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection, which offers Alexa Fluor 488, 594, and 647 fluorescent dye options. This in situ apoptosis detection kit is optimized for tissue or cell samples and the dyes can be multiplexed with other dyes or proteins, such as GFP and RFP, and incorporated more readily into complex molecules due to their smaller size (compared with antibodies). This TUNEL assay kit is also very flexible and can be used to test 1–50 samples in a single experiment.

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.

Specifications

• Description: Click-iT Plus TUNEL Assay for In Situ Apoptosis Detection, Alexa Fluor™ 647 dye
• Quantity: 1 kit
• Format: Coverslip
• Product Type: TUNEL Assay
• Excitation/Emission: 650/665
• No. of Reactions: 50 coverslips
• Color: Far-Red
• Shipping Condition: Dry Ice
• Product Line: Click-iT
• Detection Method: Fluorescence
• For Use With (Equipment): Fluorescence Microscope
• Label or Dye: Alexa Fluor™ 647
• Label Type: Alexa Fluor™ Dyes
• Storage Requirements: Store at <20°C and protect from light.

Frequently Asked Questions (FAQs)

What is the fluorescence excitation and emission maxima of Alexa Fluor 647 dye?

Alexa Fluor 647 has an excitation/emission maxima of 650/670 nm.

I will be performing a cell proliferation assay using Click-iT EdU kit. At what point can I stop overnight, or do I have to perform all the steps continuously?

One may store the sample after fixation overnight in PBS at 4^oC. For longer storage (<1 week) , store in buffer with 1-2% formaldehyde or in formalin to limit microbial growth. If you use sodium azide as a microbial inhibitor, it must be completely removed prior to the Click-iT reaction.

I need to test cells for apoptosis after they have been formaldehyde-fixed and permeabilized. What dye or conjugate do you recommend? Will Annexin V conjugates work?

We do not recommend Annexin V for post-fix labeling, since fixation inactivates the function of the translocase; fixed samples would show mostly uniform labeling with Annexin V. The only options you have for apoptosis assays after fixation are to use an anti-caspase antibody or perform a TUNEL assay, such as with the Click-iT TUNEL Imaging kits.

Can I use Click-iT Plus TUNEL Assay Kits for In Situ Apoptosis Detection (Cat. Nos. C10617, C10618, C10619) for whole mount immunofluorescence staining of zebrafish larvae?

Yes. The Click-iT Plus TUNEL Assay Kits for In Situ Apoptosis Detection (Cat. Nos. C10617, C10618, C10619) is optimized for use with tissues and should work on zebrafish larvae, although it has not been internally validated with zebrafish larvae.

I am observing no signal or very low specific signal for my click-labeled samples. What can I do to improve the signal?

The click reaction is only effective when copper is in the appropriate valency. Azides and alkynes will not react with each other without copper. Make sure that the click reaction mixture is used immediately after preparation when the copper (II) concentration is at its highest.
Do not use additive buffer that has turned yellow; it must be colorless to be active.
Cells need to be adequately fixed and permeabilized for the TdT enzyme and click reagents to have access to the nucleus. Tissue samples require digestion with proteinase K or other proteolytic enzymes for sufficient TdT access.
Some reagents can bind copper and reduce its effective concentration available to catalyze the click reaction. Do not include any metal chelator (e.g., EDTA, EGTA, citrate, etc.) in any buffer or reagent prior to the click reaction. Avoid buffers or reagents that include other metal ions that may be o xidized or reduced. It may be help to include extra wash steps on the cell or tissue sample before performing the click reaction.
You can repeat the click reaction with fresh reagents to try to improve signal. Increasing the click reaction time longer than 30 minutes will not improve a low signal. Performing a second, 30 minute incubation with fresh click reaction reagents is more effective at improving labeling.
Your cells may not be apoptotic. Prepare a DNase I-treated positive control to verify that the TdT enzymatic reaction and click labeling reaction are working correctly.

I am observing high non-specific background when I image my Click-iT EdU TUNEL-labeled samples. What is causing this and what can I do to reduce the background?

The click reaction is very selective between an azide and alkyne. No other side reactions are possible in a biological system. Any non-specific background is due to non-covalent binding of the dye to various cellular components. The Select FX Signal Enhancer is not effective at reducing non-specific charge-based binding of dyes following the click reaction; we do not recommend its use with the Click-iT detection reagents. The best method to reduce background is to increase the number of BSA washes. You should always do a no-dye or no-click reaction control under the same processing and detection conditions to verify that the background is actually due to the dye and not autofluorescence. You should also perform the complete click reaction on a no-TdT enzyme control sample to verify the specificity of the click reaction signal.

I notice that when I post-stain my cells with DAPI after performing the click reaction to detect EdU incorporation, my DAPI signal is lower compared to my no-click reaction control samples. What causes the reduction in DAPI signal?

The copper in the click reaction denatures DNA to a small extent (although not as much as is required for efficient BrdU detection), which can affect the binding affinity of DNA dyes including DAPI and Hoechst stain. This effect should only be apparent with the classic EdU kits and not the Click-iT Plus EdU kits, which use a lower copper concentration.

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.

Can I combine Click-iT EdU labeling with EdU TUNEL labeling so that I can detect proliferation and apoptosis in the same sample?

It is possible, but if you have not completely labeled all of the metabolically incorporated EdU in the first click reaction, then it will be labeled in the second click reaction for TUNEL labeling, leading to false positives for apoptotic cells. It would be simpler to combine Click-iT EdU labeling with BrdU TUNEL labeling, as BrdU detection will not cross-react with EdU labeled cells. If you really wish to perform a double EdU labeling for both proliferation and apoptosis detection, then you should repeat the click reaction to detect the metabolically incorporated EdU using fresh click reagents to ensure that all of the incorporated EdU is labeled before performing the EdU TUNEL assay. You should then perform a control no-TdT enzyme EdU TUNEL assay to verify that there is no signal generated with the TUNEL click reaction.

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.