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

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

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Screen apoptotic cells with Click-iT TUNEL Assay kits, which offer easy Alexa Fluor dye incorporation and can be multiplexed with GFP and RFP.
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Catalog NumberColorLabel or Dye
C10245GreenAlexa Fluor 488, Hoechst 33342
C10246RedAlexa Fluor 594, Hoechst 33342
C10247Far-RedAlexa Fluor 647, Hoechst 33342
Catalog number C10245
Price (USD)
1,284.65
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1,324.00
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Color:
Green
Label or Dye:
Alexa Fluor 488, Hoechst 33342
Price (USD)
1,284.65
Online Exclusive
1,324.00
Save 39.35 (3%)
Each
Add to cart
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.
For Research Use Only. Not for use in diagnostic procedures.
Specifications
ColorGreen
DescriptionClick-iT TUNEL Alexa Fluor™ 488 Imaging Assay
Excitation/Emission490/525
For Use With (Equipment)Fluorescence Microscope, High Content Instrument
Label TypeAlexa Fluor™ Dyes, Classic Dyes
Label or DyeAlexa Fluor 488, Hoechst 33342
No. of Reactions1 X 96 tests or 50 coverslips
Product LineClick-iT
Product TypeImaging Assay
Quantity1 kit
Shipping ConditionDry Ice
Storage RequirementsStore at ≤-20°C and protected from light.
Detection MethodFluorescence
Format96-well Plate
Unit SizeEach

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Labeling Chemistry Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.

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.

Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center.

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