Click-iT™ Plus TUNEL Assay Kits for In Situ Apoptosis Detection, 1 kit - FAQs

Alexa Fluor 488 has fluorescence excitation and emission maxima of 495/519 nm.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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-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.

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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.

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