Molecular Probes™ Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit

Catalog No.	Quantity
50-113-7406	50 Assays

Catalog No. 50-113-7406 Supplier Molecular Probes™ Supplier No. C10418

Description

Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit

The Click-iT™ EdU Pacific Blue™ Flow Cytometry Assay Kit provides a simplified, more robust assay for analyzing DNA replication in proliferating cells as compared to traditional BrdU methods. Newly synthesized DNA is analyzed using the 405 nm laser of the flow cytometer.

Accurate—superior results compared to BrdU assays

Fast—results in as little as 90 minutes

Economical—more assays per kit

View selection guide for all Click-iT™ EdU and Click-iT™ Plus EdU assays for flow cytometry.

An Advanced Method Giving You Results Superior to BrdU
The most accurate method of proliferation analysis is direct measurement of DNA synthesis. Originally, this was performed through incorporation of radioactive nucleosides, i.e., ³H-thymidine. This method was replaced by antibody-based detection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT™ EdU Flow Cytometry Assay Kits are novel alternatives to the BrdU assay. EdU (5-ethynyl-2´-deoxyuridine) is a thymidine analog which is incorporated into DNA during active DNA synthesis. Detection is based on click chemistry: a copper catalyzed covalent reaction between an azide and an alkyne. In this application, the alkyne is found in the ethynyl moiety of EdU, while the azide is coupled to Alexa Fluor ™ 488, Alexa Fluor™ 647, or Pacific Blue™ dyes. Standard flow cytometry methods are used for determining the percentage of S-phase cells in the population (Fig 1).

Mild Conditions Allow Use with Cell Cycle Dyes and Antibodies
The advantages of Click-iT™ EdU labeling are readily evident while performing the assay (Fig 2). The small size of the dye azide allows for efficient detection of the incorporated EdU using mild conditions, while standard aldehyde-based fixation and detergent permeabilization are sufficient for the Click-iT™ detection reagent to gain access to the DNA. This is in contrast to BrdU assays that require DNA denaturation (using HCl, heat, or digestion with DNase) to expose the BrdU so that it may be detected with an anti-BrdU antibody. Sample processing for the BrdU assay can result in signal alteration of the cell cycle distribution as well as destruction of antigen recognition sites when using the HCl method. In contrast, the easy-to-use EdU cell proliferation kit is compatible with cell cycle dyes. This EdU assay kit can also be multiplexed with antibodies against surface and intracellular markers.

Quick and Simple Protocol
The Click-iT™ EdU protocol is based on the aldehyde fixation and detergent permeabilization steps for immunohistochemical antibody labeling, but EdU is compatible with other fixation/permeabilization agents including saponin and methanol. In just five steps you’ll be ready to analyze your cell proliferation data:

Treat cells with EdUM

Fix and permeabilize cells

Detect S-phase cells with Click-iT™ detection cocktail for 30 min

Wash once

Analyze

Get Accurate Results Economically
By increasing the number of assays per kit, the Click-IT™ EdU Pacific Blue™ Flow Cytometry Assay Kit is less expensive than the traditional BrdU assays making them ideal for large experiments.

For Research Use Only. Not for use in diagnostic procedures.

Specifications

• Content And Storage: Contains EdU (5-ethynyl-2' -deoxyuridine), Pacific Blue™ azide, anhydrous dimethylsulfoxide (DMSO), Click-iT™ fixative, Click-iT™ saponin-based permeabilization and wash buffer, copper (II) sulfate,, and Click-iT™ EdU buffer additive.
  Store at 2°C to 8°C
  Dessicate and protect from light.
• Detection Method: Fluorescence
• For Use With (Equipment): Flow Cytometer
• Product Type: Flow Cytometry Assay Kit
• Dye Type: Pacific Blue™
• Emission: 404/450
• Format: Tube(s)
• Product Line: Click-iT, Pacific Blue
• Quantity: 50 Assays
• Shipping Condition: Room Temperature

Frequently Asked Questions (FAQs)

What are the main characteristics of a Click-iT reaction?

Click reactions have several general characteristics: the reaction is efficient, no extreme temperatures or solvents are required, the reaction is complete within 30 minutes, the components of the reaction are bio-inert, and perhaps most importantly, no side reactions occur-the label and detection tags react selectively and specifically with one another. This final point is a key advantage of this powerful detection technique; it is possible to apply click chemistry-labeled molecules to complex biological samples and detect them with unprecedented sensitivity due to the extremely low background of the reaction.

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?

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.

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.

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 am observing no signal or very low 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. 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.

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.

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.

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.

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

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.

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

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.

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?

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.

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

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.

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

For Research Use Only. Not for use in diagnostic procedures.