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View additional product information for Alexa Fluor™ 647 Azide, Triethylammonium Salt - FAQs (A10277)
16 product FAQs found
Yes. You can purchase Alexa Fluor 647 Azide, Triethylammonium Salt (Cat. No. A10277).
Unfortunately, the actual concentration of the azide dyes used in the Click-iT assay kits is proprietary. However, we recommend making up a 1-10 mM azide stock solution in high-quality anhydrous DMSO and using it at approximately 1-5 µM final concentration in the click reaction. You will need to work out a dye concentration empirically. Too high of a dye concentration can result in high non-specific background or dye aggregates, whereas too low of a concentration may not give a strong enough signal.
Find additional tips, troubleshooting help, and resources within our Cell Viability, Proliferation, Cryopreservation, and Apoptosis Support Center.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Yes, EdU is frequently used for in vivo assays and acceptable EdU incorporation can be obtained following injection or media incubation. Optimal EdU incubation times and concentrations will depend on the incorporation method and the organism/tissue type. Recommendations on EdU incorporation methods in various model organisms and protocols for detection of EdU in tissue samples can be found in the In Vivo Use of Click-iT EdU Cell Proliferation Assays application note and the Click-iT EdU Labeling In Vivo Cell Proliferation Protocol.
Table 2 of the EdU (5-ethynyl-2'-deoxyuridine) product manual also provides some recommendations. Methods that have been optimized for BrdU labeling for your organism are a good starting point. Tissue samples can be frozen or paraffin-embedded and then processed using normal procedures for subsequent IHC staining. For detection of EdU, use any desired method for fixation and permeabilization, and then perform the click detection reaction exactly according to the protocol for cultured cells in the product manual.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
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.
The Click-iT EdU flow kits use a lower azide dye concentration than what is optimal for the EdU imaging kits, so it is not possible to use a flow kit to label cells for an imaging experiment. It is possible to use a Click-iT EdU imaging kit for samples for flow analysis, but it would be necessary to optimize the azide dye concentration first. The azide dye concentrations used in the Click-iT EdU kits are proprietary.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
The copper sulfate solution component supplied in the Click-iT kits is 100 mM copper sulfate in water. Copper (II) sulfate (CuSO4) powder is available from many chemical suppliers.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
Unfortunately, the actual concentration of the azide dyes used in the Click-iT assay kits is proprietary, so we cannot provide the exact concentration used. For a general guideline, we recommend making up a 1-10 mM DMSO stock solution and using at approximately 1-10 µM final concentration in the click reaction. Too high of a dye concentration can result in high nonspecific background or dye aggregates; too low of a concentration may not give a strong enough signal.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.
If you are using EdU or EU, the simplest thing to do is purchase a complete kit that is specific for the type of assay you are performing, either flow, imaging, or a microplate assay. The complete kits contain the azide detection reagent as well as copper and the buffers necessary to perform the click reaction. If you have a different alkyne metabolite analog or do not wish to purchase EdU or EU, then you will need to purchase any of our azide dye detection reagents and the Click-iT Cell Reaction Buffer Kit (Cat. No. C10269), which contains the copper solution and the other buffer reagents necessary to perform the click reaction for flow and imaging assays. If you wish to perform a microplate assay, then you are limited to the Click-iT EdU Microplate Assay (Cat. No. C10214), which provides all the reagents necessary for the click reaction as well as the reagents necessary to perform the amplification of the signal so that it can be detected on a microplate reader.
Find additional tips, troubleshooting help, and resources within our Cell Analysis Support Center.