Q: A control for a Click-iT EdU labeling experiment uses no EdU and the Click-iT reaction using Alexa Fluor 594 azide. The mouse heart tissue sections are showing non-specific labeling in red, seen in particular clusters of cells. They don't overlap with DAPI. What is the problem?

The problem is likely not the Alexa Fluor 594 azide. Since there are no alkynes endogenous to mouse tissue, there is nothing for the dye-azide to bind to. Since the background doesn't overlap with nuclei (DAPI signal), this isn't an issue of unintended EdU labeling. This red is autofluorescence from red blood cells; they autofluoresce in the red and don't have nuclei. This can be confirmed by checking a completely unlabeled tissue section (no dye present at all) to see if they are still present and by examining the cells at high magnification and looking for corpuscular shape. Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center .

Q: Can I combine Click-iT or Click-iT Plus reactions with phalloidin conjugates used for actin staining?

We do not recommend using phalloidin conjugates for staining actin in combination with traditional Click-iT or Click-iT Plus reactions since phalloidin is extremely sensitive to the presence of copper. For staining actin in combination with traditional Click-iT or Click-iT Plus reactions, we recommend using anti-α-actin antibodies for staining actin in the cytoskeleton. You can find a list of our actin antibodies here . Another option would be to use the Click-iT Plus Alexa Fluor Picolyl Azide Toolkit (Cat. Nos. C10641, C10642, C10643). These Click-iT Plus toolkits provide Copper and Copper protectant separately which makes it easier to titrate the copper concentration to obtain optimal labeling with minimal copper-mediated damage. You may need to optimize the click reaction with the lowest possible concentration of copper and then perform the phalloidin staining. Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center .

Q: Are the Alexa Fluor azides from Click-iT EdU kits available separately?

Yes, but the standalone products are not shipped at the same amount as provided in the Click-iT EdU kits; the amount of dye-azide provided in the Click-iT kits is proprietary information. See these catalog numbers for the standalone products: - Cat. No. A10266 : Alexa Fluor 488 azide - Cat. No. A10270 : Alexa Fluor 594 azide - Cat. No. A10277 : Alexa Fluor 647 azide Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center .

Q: 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 .

Q: 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 .

Q: 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 .

Q: I am observing high background when I analyze my click-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 can also perform the complete click reaction on a carrier solvent-only, no EdU or no-EU control to verify the specificity of the click reaction signal. Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center .

Q: Can I perform Click-iT EdU detection on live cells?

No, the EdU metabolic labeling reagent must be used on live cells, but the actual click detection reaction must be performed on fixed and permeabilized samples, as the azide detection reagents and buffer components are cell impermeant. Find additional tips, troubleshooting help, and resources within our Flow Cytometry Support Center .

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

Question 1

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?

Accepted Answer

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.

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

Question 2

A control for a Click-iT EdU labeling experiment uses no EdU and the Click-iT reaction using Alexa Fluor 594 azide.  The mouse heart tissue sections are showing non-specific labeling in red, seen in particular clusters of cells.  They don't overlap with DAPI. What is the problem?

Accepted Answer

The problem is likely not the Alexa Fluor 594 azide. Since there are no alkynes endogenous to mouse tissue, there is nothing for the dye-azide to bind to. Since the background doesn't overlap with nuclei (DAPI signal), this isn't an issue of unintended EdU labeling. This red is autofluorescence from red blood cells; they autofluoresce in the red and don't have nuclei. This can be confirmed by checking a completely unlabeled tissue section (no dye present at all) to see if they are still present and by examining the cells at high magnification and looking for corpuscular shape.

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

Question 3

Can I combine Click-iT or Click-iT Plus reactions with phalloidin conjugates used for actin staining?

Accepted Answer

We do not recommend using phalloidin conjugates for staining actin in combination with traditional Click-iT or Click-iT Plus reactions since phalloidin is extremely sensitive to the presence of copper.

For staining actin in combination with traditional Click-iT or Click-iT Plus reactions, we recommend using anti-α-actin antibodies for staining actin in the cytoskeleton. You can find a list of our actin antibodies here.

Another option would be to use the Click-iT Plus Alexa Fluor Picolyl Azide Toolkit (Cat. Nos. C10641, C10642, C10643). These Click-iT Plus toolkits provide Copper and Copper protectant separately which makes it easier to titrate the copper concentration to obtain optimal labeling with minimal copper-mediated damage. You may need to optimize the click reaction with the lowest possible concentration of copper and then perform the phalloidin staining.

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

Question 4

Are the Alexa Fluor azides from Click-iT EdU kits available separately?

Accepted Answer

Yes, but the standalone products are not shipped at the same amount as provided in the Click-iT EdU kits; the amount of dye-azide provided in the Click-iT kits is proprietary information. See these catalog numbers for the standalone products:
- Cat. No. A10266: Alexa Fluor 488 azide
- Cat. No. A10270: Alexa Fluor 594 azide
- Cat. No. A10277: Alexa Fluor 647 azide

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

Question 5

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

Accepted Answer

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.

Question 6

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?

Accepted Answer

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.

Question 7

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?

Accepted Answer

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.

Question 8

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

Accepted Answer

The click reaction is only effective when copper is in the appropriate valency. Except for the DIBO alkyne-azide reaction, 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 click reagents to have access to intracellular components that have incorporated the click substrate(s).
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 oxidized 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.
Low signal can also be due to low incorporation of EdU, EU, or other click substrates. Other click substrates (e.g., AHA, HPG, palmitic acid, azide, etc.) incorporated into cellular components may have been lost if not adequately cross-linked in place or if the wrong fixative was used. For click substrates that are incorporated into the membrane or lipids, you should avoid the use of alcohol or acetone fixatives and permeabilizing agents.
The incorporated click substrate must be accessible at the time of the click reaction; labeling of incorporated amino acid analogs may be lower in native proteins relative to denatured proteins.
You may need to optimize the metabolic labeling conditions including analog incubation time or concentration. Cells that are healthy, not too high of a passage number and not too crowded may incorporate the analog better. You may create a positive control by including extra doses of the click substrate during multiple time points during an incubation time that spans or closely spans the doubling time of the cell type of interest.

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

Question 9

I am observing high background when I analyze my click-labeled samples. What is causing this and what can I do to reduce the background?

Accepted Answer

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 can also perform the complete click reaction on a carrier solvent-only, no EdU or no-EU control to verify the specificity of the click reaction signal.

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

Question 10

Can I perform Click-iT EdU detection on live cells?

Accepted Answer

No, the EdU metabolic labeling reagent must be used on live cells, but the actual click detection reaction must be performed on fixed and permeabilized samples, as the azide detection reagents and buffer components are cell impermeant.

Find additional tips, troubleshooting help, and resources within our Flow Cytometry Support Center.

Question 11

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

Accepted Answer

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.

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

Question 12

Can I combine EdU and BrdU labeling and detection on the same sample?

Accepted Answer

Yes, EdU and BrdU labeling can be combined for dual-pulse labeling of cell proliferation in cultured cells and in vivo. BrdU will be preferentially incorporated into DNA, so perform the EdU incubation first followed by the BrdU incubation. Removal of EdU from the media is not required in cultured cells when BrdU is added as the second label. Perform an alcohol fixation followed by some method of DNA denaturation as required for the BrdU detection protocol and then perform the click labeling reaction for detection of EdU followed by antibody labeling for detection of BrdU. Be sure to select a BrdU antibody that does not have cross-reactivity to EdU, such as our MoBU-1 clone (Cat. No. B35141). Many BrdU antibodies have been shown to have some amount of cross-reactivity with incorporated EdU. Here is a link (http://www.thermofisher.com/us/en/home/references/protocols/cell-and-tissue-analysis/flow-cytometry-protocol/cell-proliferation/dual-pulse-labeling-of-cell-proliferation-using-edu-and-brdu-incorporation.html) to an example protocol for dual-pulse labeling using EdU and BrdU.

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

Question 13

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

Accepted Answer

We have not validated the use of EdU for proliferation 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.

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

Question 14

I am collecting samples over time and would like to perform the Click-iT detection reaction on all the samples at the same time. Are there stopping points in the protocol, so that I do not have to perform the entire detection procedure in the same day?

Accepted Answer

Yes, you can store samples after fixing in formaldehyde and washing, before the permeabilization step. Just keep the cells in PBS, cover and seal the container well, and store at 4 degrees C. The cells should be fine for at least a week. You can also store the samples after the click reaction and wash steps and then perform any immunostaining and nuclear counterstaining on the following day.

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

Question 15

Can I substitute reagents from the Click-iT Plus kits into the original Click-iT kits or vice versa?

Accepted Answer

No, the detection reagent and reagents necessary to perform the click reaction cannot be intermixed between the Click-iT Plus and original Click-iT kits. The Click-iT Plus assay uses a modified picolyl azide dye and reduced copper concentration combined with a special copper protectant that localizes the copper at the click reaction, while the original Click-iT kits use an unmodified azide dye and higher copper concentrations to perform the click reaction.

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

Question 16

What is the difference between the Click-iT Plus and the original Click-iT assay kits?

Accepted Answer

The Click-iT Plus assay uses a modified picolyl azide dye and reduced copper concentration combined with a special copper protectant that localizes the copper at the incorporated alkyne group and thus minimizes copper damage to biomolecules. The original Click-iT kits use an unmodified azide dye and higher copper concentrations to perform the click reaction, which may inactivate enzymes, including HRP, and will quench the fluorescence of GFP, RFP, mCherry and other fluorescent proteins, as well as R-phycoerythrin. If you do not wish to modify your antibody staining protocol or have fluorescent protein-expressing cells, then use the Click-iT Plus kits.

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

Question 17

What are the advantages of flow cytometry?

Accepted Answer

-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.

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

Question 18

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

Accepted Answer

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.

Find additional tips, troubleshooting help, and resources within our Flow Cytometry Support Center.

Question 19

Can any type of cell incorporate EdU?

Accepted Answer

No. The cell must possess a pyrimidine salvage pathway—without this pathway, EdU does not become phosphorylated to allow incorporation into replicating DNA.

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

Question 20

In the Click-iT EdU kits, why is 1% or 3% BSA included in the PBS wash buffers after fixation?

Accepted Answer

In the wash step after fixation, BSA quenches any unreacted formaldehyde.

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

Click-iT™ EdU Cell Proliferation Kit for Imaging, Alexa Fluor™ 488 dye - FAQs