Invitrogen™ Click-iT™ TUNEL Alexa Fluor Imaging Assays for Microscopy & HCS

Catalog No.	Color	Label or Dye
C10247	Far-Red	Alexa Fluor 647, Hoechst 33342
C10246	Red	Alexa Fluor 594, Hoechst 33342
C10245	Green	Alexa Fluor 488, Hoechst 33342

Catalog No. C10247 Supplier Invitrogen™ Supplier No. C10247

Description

Includes

One kit containing sufficient reagents to perform 1 x 96 tests or 50 coverslips.

Screen apoptotic cells with Click-iT TUNEL Assay kits, which offer easy Alexa Fluor dye incorporation and can be multiplexed with GFP and RFP.

Screen apoptotic cells easily and efficiently in just two hours with the Click-iT TUNEL Alexa Fluor 488, 594, and 647 imaging assays. Intended for use with microscopy and high-content screening (HCS), these TUNEL assay kits use a copper-catalyzed reaction to detect incorporated alkyne-modified dUTPs on terminal DNA ends of fragmented DNA. Compared with assays using other modified nucleotides, these apoptosis detection kits are fast (complete within two hours) and can detect a higher percentage of apoptotic cells under identical conditions. Click-iT TUNEL assays also allow multiplexing with surface and intracellular biomarker detection methods, including those that measure fluorescent signal from GFP or RFP.

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.

Order Info

Shipping Condition: Dry ice

Specifications

• Description: Click-iT TUNEL Alexa Fluor™ 647 Imaging Assay
• Quantity: 1 kit
• Format: 96-well Plate
• Product Type: Imaging Assay
• Excitation/Emission: 650/665
• No. of Reactions: 1 X 96 tests or 50 coverslips
• Color: Far-Red
• Shipping Condition: Dry Ice
• Product Line: Click-iT
• Detection Method: Fluorescence
• For Use With (Equipment): Fluorescence Microscope, High Content Instrument
• Label or Dye: Alexa Fluor 647, Hoechst 33342
• Label Type: Alexa Fluor™ Dyes, Classic Dyes
• Storage Requirements: Store at ≤-20°C and protected from light.

Frequently Asked Questions (FAQs)

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 TUNEL Alexa Fluor Imaging Assays for Microscopy & HCS (Cat. No. C10246) for flow cytometry?

We have not validated the use of Click‐iT TUNEL assay for flow cytometry. Theoretically, any Click‐iT TUNEL assay for imaging can be adapted to be used with flow cytometry. In general, follow the protocol as provided but spin down the suspension cells after every step. Start with about 10∧6 cells at about 10∧7 cell/mL. Please note that flow cytometry is more sensitive than fluorescence imaging, so you should use between 1/5th to 1/10th of the azide dye detection reagent in the click reaction. All other concentrations of the click reaction reagents should stay the same. We recommend using the Click‐iT Plus TUNEL assays (C10617, C10618, C10619), as the detection reagent is provided in a separate vial, enabling you to modify the concentration used. The Click‐iT Plus TUNEL assay protocol can be found on the following link.

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.