We recently developed our own EdU labeling approach using the following cocktail, adding reagents in the order listed:

This cocktail works well for EdU development in both cells (4% PFA fixed) and mouse brain tissue (4% PFA or PLP-fixed, 40 um thick sections). Our general protocol is the following. We complete the EdU development before blocking as normal for subsequent antibody-based labeling of other antigens.

We also tested sulfonated vs. non-sulfonated fluorophore-azides and found that the sulfonated versions are necessary to avoid general tissue staining in aqueous buffers. We could destain cells with ethanol but not sufficiently destain tissue with the non-sulfonated azides. Lumiprobe currently has sulfo-Cy3/5/7 azides available. We reconstitute these azides in water to create 2 mM stocks. These can be used in aqueous buffers without organic solvent and don't require any special destaining.

Our lab has been using this approach to develop EdU in cells pulsed with 10 uM EdU (minutes to hours) and in tissue from either mice injected i.p. with 25 mg/kg EdU or mice that received EdU in drinking water at 200 ug/ml for 1 week. We use Edu from Carbosynth as it is considerably cheaper than from other vendors. 

We have not found TBTA to be necessary for these specific applications. Even though Tris buffers slow the CuAAC reaction, we've found they work well here. I believe that a Tris buffer is the one supplied in commercial labeling kits.

Hope this helps everyone!

http://www.lumiprobe.com/catalog/dye-azides

http://www.carbosynth.com/carbosynth/website.nsf/(w-productdisplay)/D1C8F04435B507E2802574E6002AA46A

Yes, we use a "homemade" recipe do to click reactions. You can easily find out the composition of click it buffer and concentration of CuSo4 used. We do it and get wonderful results. I think its citrate buffer and 10M CuSO4 but not sure. I am traveling now so can't confirm. But if you want I can ask my colleague to post it here.

Update:

I found the following reagent at nearly 1/10th the cost of AlexaFluor 647 Azide:

http://www.lumiprobe.com/p/sulfo-cy5-azide

I haven't used it yet, so I don't know how well it works, but looks promising.  It has the sulfonation to improve solubility and reduce aggregation, along with the click azide group.  Does anyone have experience with this reagent?

We've tackled this problem by making our own buffers for Click-it chemistry, and by reducing the amount of EdU using for labeling (you can try 1:1000 to 1:5000 dilution range). In addition, we purchased the AF488-azide from Invitrogen in bulk as a custom synthesis order, which also saves on cost relative to the individual kits. Haven't used the Lumiprobe reagent, but this might be another viable option for cost savings.

@Lale, I've been trying to optimize BrdU staining and, whereas it does work, the signal-noise is much lower than EdU-click staining.  Additionally, I am constrained to DNase antigen retrieval, since I have fluorescent proteins in my sample I need to preserve.  This of course makes nuclear counterstaining difficult.  With the low signal from both BrdU and nuclear counterstain, it is difficult to distinguish real signal against the high background in primary cardiomyocytes.

@Daniel @Sanket

I would be grateful for your buffer recipes/protocol.

So far, I have drawn up the following (based mostly on Salic & Mitchison 2008, PNAS).  Please let me know if you have any suggestions/words of caution:

Dissolve fluorophore-azide in DMSO to 10 mM

After fixation/Permeabilization of sample, prepare:

     700 ul of 143 mM Tris, pH 8.5 (Final: 100 mM)

     1 ul of fluorophore-azide (Final: 10 uM)

     100 ul of 10 mM CuSO4 (Final: 1 mM)

     200 ul of 500 mM Ascorbic acid* (Final: 100 mM)

     Mix and add immediately to sample

Incubate 30 min

Wash 3X with PBS

Nuclear counterstain/Ab stain if needed

Image

*Note, it was not explicitly stated that the pH of the ascorbic acid was adjusted, does anyone know how this will affect the reaction?

I have been researching click chemistry reactions (not EdU) and I think i came up with something similar. One difference is the use of cofactor in the reaction. For fixed cells/tissue is TBTA (678937 sigma) but can be other if is not fixed tissue/cells (THPTA or BTTAA). From Hong et al.,  (2009) "THPTA serves the dual purpose of protecting biomolecules from hydrolysis by Cu(II) byproducts, and sacrificially intercepting the radicals and/or peroxides derived from O2/Cu/ascorbate that oxidize histidine and other residues. An excess of ligand does not dramatically slow the reaction, so you can use more than 5 equivalents if needed".

also, the ascorbic solution is 100mM for the stock. the protocol I found calls for 2.5 mM as final concentration (have seen 5 mM).  This solution must be made fresh. Got it form this paper (Uttamapinant et al., 2013. Nature protocols). 

Also tris buffer is not recommended (see Presolski et al., 20011).  

Update: I performed the protocol I posted above, except at pH 8 and incubated overnight.  It worked a lot better than my best attempt at BrdU staining, even with the Tris and without a cofactor (such as TBTA).  I'm sure it could get better with optimization.

Glad to hear it is working, Justin. Thanks for posting the update- very useful information for anyone checking your post.

Maybe these? 

http://www.emdmillipore.com/Web-US-Site/en_CA/-/USD/ViewParametricSearch-SimpleOfferSearch?SynchronizerToken=7df2522071653541be3cb61dd70f66b89d88feefd97c20b3cdede8664937dc8c&SynchronizerToken=7df2522071653541be3cb61dd70f66b89d88feefd97c20b3cdede8664937dc8c&search=&TrackingSearchType=SB+-+Search+Box&SearchContextPageletUUID=&SearchTerm=edu

Hello everyone! I'm very glad that this discussion was running. Justin, is it possible that you have your protocol optimized and maybe published somewhere in the methods section? It would be a huge help. Thanks!

We recently developed our own EdU labeling approach using the following cocktail, adding reagents in the order listed:

This cocktail works well for EdU development in both cells (4% PFA fixed) and mouse brain tissue (4% PFA or PLP-fixed, 40 um thick sections). Our general protocol is the following. We complete the EdU development before blocking as normal for subsequent antibody-based labeling of other antigens.

We also tested sulfonated vs. non-sulfonated fluorophore-azides and found that the sulfonated versions are necessary to avoid general tissue staining in aqueous buffers. We could destain cells with ethanol but not sufficiently destain tissue with the non-sulfonated azides. Lumiprobe currently has sulfo-Cy3/5/7 azides available. We reconstitute these azides in water to create 2 mM stocks. These can be used in aqueous buffers without organic solvent and don't require any special destaining.

Our lab has been using this approach to develop EdU in cells pulsed with 10 uM EdU (minutes to hours) and in tissue from either mice injected i.p. with 25 mg/kg EdU or mice that received EdU in drinking water at 200 ug/ml for 1 week. We use Edu from Carbosynth as it is considerably cheaper than from other vendors. 

We have not found TBTA to be necessary for these specific applications. Even though Tris buffers slow the CuAAC reaction, we've found they work well here. I believe that a Tris buffer is the one supplied in commercial labeling kits.

Hope this helps everyone!

http://www.lumiprobe.com/catalog/dye-azides

http://www.carbosynth.com/carbosynth/website.nsf/(w-productdisplay)/D1C8F04435B507E2802574E6002AA46A

Hello! I am also trying to detect EdU (on sections)  trough ‘click it reaction’ using a very similar protocol. When I add copper to the reaction cocktail (100mM Tris pH 7.6, 1mM copper(II)-TBTA, 10uM FAM-azide 6-isomer, 100mM ascorbic acid) the solution gets white, opaque and forms very small precipitates that impairs further analysis. Did anyone had the same problem? Or any idea how to fix it?

Thanks in advance!

Joana, have you tried adding the copper to your Tris buffer first, then adding the azide and ascorbic acid? We have had similar issues when the ascorbic acid was present in the buffer before adding copper or azide. 

Hi Andrew, thanks for your reply! The order of addition that I performed was actually that, first adding copper to the Tris and afterwards azide and ascorbic acid. As soon as I had copper to the solution (so, only Tris and copper present) the solution gets white precipitates. I am using Copper(II)-TBTA from lumiprobe. I think that  the pH of Tris may influence this change in the solution? Do you have any idea?

Hi Joana! If you are using a non-sulfonated FAM azide, I suspect that the azide may be the issue. Is your 10 uM FAM azide prepared in organic solvent? Even if it is, you may need to prepare the entire cocktail with at least 20% DMF or DMSO to improve solubility and avoid having it precipitate in your aqueous solution. If you can't use that much organic co-solvent in your application, try switching to a sulfonated azide. Hope that you figure this out!

Thanks for your suggestions. Indeed I am using a non-sulfonated FAM azide, but the cocktail had already precipitates even before adding the azide. The azide is dissolved in DMSO but I will then try to prepare the cocktail with DMSO. Hopefully soon I will have an nice update. 

Hi all. really interesting reading all your protocols. Has anyone tried diluting the components of the life technologies EdU click it kit any further than is suggested (purely for making it go further!)?

Hi Justin. You can try baseclick EdU kits (see webpage www.baseclick.eu). Those kits have very similar or even superior performances. baseclick offers also other fluorescent dyes along with the standars used from other companies. The price is lower due to the simple fact that baseclick owns the IP. The kits are made in Germany and the company is ISO9001 certified. The shipping cost are below $50 for US...and I'm sure you can get also a discount ;)

http://www.baseclick.eu/products.php?IDCAT=24&IDSOTTOCAT=25

Hi all. I do a lot of EdU click reaction for detection in tissues (both normal and cancerous- tried in gliomas and breast cancer), and in in vitro cells. I simply resuspend all the components (copper sulphate, sodium ascorbate, fluorophore azide) in PBS. Works extremely well and I could even isolate RNA from click reacted cells. The only expensive thing I have to order is the fluorophore azide. Hope this helps everyone save cost.

Berwini, can you specify concentrations/ volumes you're using in your case? We've now tried it with triethylammonium acetate (TEAA) buffer (pH7), but it seems to be working less bright (efficient) than the kit. The concentrations are as follows:

Final conc. per 100ul staining volume

TEAA  - 1M, pH 7 

DMSO - 50% of the total 

CuSO4 - 1mM

Azide - 5uM 

Ascorbic acid - 10mM

We've previously tried it with Tris buffer, and the reaction failed.

Hi Ieva. Per ml of reaction mixture in PBS, I use 10 uM of fluorophore azide, 1.5 mM CuSO4, and 20 mg/ml sodium ascorbate. Hope this helps. 

Dear Ieva, I have used 0.05 M Tris and 0.15 M NaCl pH 7,5 as basic buffer (as Andrew Brumm suggested) and it worked very well.

 I'm interested in using Edu to measure T cell proliferation in mouse brains. Andrew, I noticed that you mentioned using Edu injections in mice. Do you have a publication, or protocol you would mind sharing?

Hi Chandra. We do EdU injections in mice to look at proliferation and just do i.p injections of 25mg/kg with a 4 hour chase. We use the EdU from life technologies but I imagine the EdU from elsewhere will be the same. Hope this helps!

We have recently set-up the EdU-click chemistry staining, based on the protocol of Andrew Brumm. I want to share our working protocol (it is quicker, more robust and brighter than our previous BrdU/Antibody labeling) which we use for teaching 2nd year bachelor students in a molecular cell biology course. We use HeLa cells grown on 12 mm coverslips.

======================================

Reagents for Click Chemistry:

100 mg EdU (5-ethynyl-2'-deoxyuridine) was from lumiprobe (10540)

5 mg Sulfo-Cyanine3 azide was from lumiprobe (B1330)

25 g L-Ascorbic acid was from Sigma (A4544)

100 g Copper(II) sulfate pentahydrate was from Sigma (12849)

======================================

Stock solutions:

EdU: 5 mM in H2O (12.5 mg in 10 ml) (final 20 µM)

Sulfo-Cy3-Azide: 4 mM in H2O (3 mg in 1 ml H2O) (final 8 µM)

CuSO4.5H2O: 200 mM in H2O (250 mg in 5 ml) (final 2 mM)

Ascorbic acid 200 mg/ml (FRESH!!) (final 20 mg/ml)

Make label mix just before use by combining (in this order; precipitate is formed after addition of CuSO4 to PBS, this is dissolved after addition of ascorbate):

PBS 888 µl

CuSO4 10 µl

Azide-dye 2 µl

Ascorbate 100 µl

======================================

Protocol

Add 4 µl of 5 mM Edu to 1 ml of complete growth medium (DMEM, FCS, P/S), final concentration is 20 µM.

Incubate cells for 30 min.

Wash 1x PBS

Fix 10 min. with 4% Formaldehyde in PBS

Incubate 5 min in 100 mM Tris

Permeabilize 10 min in PBS + 0.1% Triton X-100

Wash 3x with PBS

Prepare label mix

Incubate cells 30 min. with label mix (typically 50 µl for a 12 mm diameter coverslip, or 200 µl for a 24 mm diameter coverslip)

Wash cells 3x 5 minutes with PBS.

Incubate cells 5 minutes with PBS with 0.1 µg/ml DAPI

Wash 1x with PBS

Mount (Mowiol)

We also had good results when DAPI was included in the Mowiol

Thanks everybody for the nice discussion. Have any of you used Click-it EdU labeling kit from Invitrogen for Double staining for glial cells? please let me know if you have got reliable results.  

Asghar - We have used the Click-It Edu kit from Invitrogen (Thermo). After the Click reaction, we have done Hoescht 33342 staining and standard immunocytochemistry without any issues. We have also had dyes like MitoTracker Red survive the Click-It reaction.

We are trying our own home made version of the kit use Atto-647N Azide from Sigma. I will have to check the Ascobic acid concetration used, and we are using 100 mM Tris / 110 mM NaCl, pH 8.5 final. Staining is not quite as bright as the Kit, but it is good enough. Also, we note little difference between 10 and  30 µM Azide. Fluorophore usage, which is a breakpoint for us between the kit or homemade version being cheaper.

You shouldn't use Tris as a buffer during the click reaction. It has been shown to inhibit it, because the free amine coordinates to the Copper. I suggest using a HEPES or phosphate buffer (pH 7.4) instead. Also, you can add 5 uM of this ligand to stabilize the copper: THPTA: Sigma-Aldrich: 762342

Very helpful discussion, thanks all!

I've recently started using the Invitrogen Click-it kit for labeling proliferating splenic germinal center B cells to great effect. EdU was given by i.p. injection. Now I would like to try something a bit different: stable labeling of plasma cells and memory B cells that emerge from the germinal center response (upon differentiation, plasma and memory become quiescent). Ideally, EdU needs to be bioavailable for at least 7 days, so instead of injecting I would like to provide it in drinking water. Has anyone had success labeling proliferating splenocytes by giving EdU through drinking water? I've seen 200 ug/mL and 1 mg/mL, but both were for different tissues. 

Any advice would be greatly appreciated! If someone has successfully achieved this kind of labeling in spleen, your protocol would be very helpful. Importantly, please indicate where EdU was purchased, based on above advice I plan to try Carbosynth EdU and follow up with Click-iT kit reagents.

Does anyone know if these "home-made" protocols also affect PE negatively?

Hello, I have a question to add to this:  

I've been using Dr. Brumm and Dr. Goedhart's protocols (Thank you!) for a 96-well format.

The staining works great, but is barely detectable on a plate reader even with 60% cells being Edu positive.  I think the plate reader may have trouble detecting it since all the fluorescence is flat on the bottom (in fixed cells) rather than in suspension.

Has anyone tried lysing the stained cells in a way that preserves fluorescence to improve signal detection?

There is one more suggestion related to “home-made” Click-iT Plus EdU Imaging Kit, which utilizes a more reactive version of azides – picolyl azides. Here is a link to flyer with some useful description and references (https://clickchemistrytools.com/wp-content/uploads/2016/11/Picolyl-azide-flyer.pdf)

We also offer a list of fluorescent picolyl azides that can be used instead of fluorescent picolyl azide provided in these kits. (https://clickchemistrytools.com/product-category/click-chemistry-toolbox/click-chemistry-toolbox-azide-reagents/click-chemistry-toolbox-azide-reagents-fluorescent-picolyl-azides/)

Hey  everyone thanks for the great tips!

I just wanted to know if someone used Joachim Goedhart  protocol for flowcytometery?

We do use very similar protocol for flowcytometry and works very well. By the way, we would like to do "home-made" Click-iT EdU assay with Pacific blue range fluorophore-azides. But as far as I know there is no sulfonated azide for it. So we tried non-sulfonated azide. It had very high background and I wish I read Dr. Brumm's comment before we tried. Should I wash with 100% ethanol to destain cells? The paper that I read used Triton or Tween 20 to wash cells after the EdU staining. I used saponin-based permeabilization and wash reagent from Invitrogen EdU kit.

In case anybody is still interested in homemade Click-It Edu kits and wants to use the same Alexa dyes as in original kits, a full like of Alexa Flour Azide (AF Azide) are available at the fraction of Thermo Fisher's price.

Here is a link to it: https://clickchemistrytools.com/product-category/click-chemistry-toolbox/click-chemistry-toolbox-azide-reagents/af-dyes/

Edu and 5-EU is also available at bulk pricing :

https://clickchemistrytools.com/product-category/metabolic-labeling-reagents/

Hi,

Has anyone used a homemade kit with Picolyl azides? I am planning on staining brain cryosections (fixed in 4% PFA) and I am unsure if the use of a picolyl azide would change the protocols you all listed above. We are staining a lot of tissue so I am thinking the homemade kit would be the most cost effective route.

Regarding the copper protectant, a TBTA protectant is best for PFA fixed tissue? Elsewhere I saw that it is used at a 1:1 with the CuSO4, does this seem appropriate?

Michelle, TBTA is not the best ligand for the reaction with picolyl azides. BTTAA and THPTA seems to be the best option for staining with picolyl aizdes. As for the protocol changes you might need to lower copper concentration and picolyl azide concentration. It helps minimize background signal without loosing any staining intensity. Here is few links to papers utilizing picolyl azides

Article Fast, Cell-Compatible Click Chemistry with Copper-Chelating ...

Article Monitoring Dynamic Glycosylation in Vivo Using Supersensitiv...

http://www.jlr.org/content/early/2016/08/26/jlr.D070565.1.short

Did anybody ever try to add the DAPI counterstain directly to the click chemistry label mix? Would it be a viable option to save time by combining both EdU detection and Nuclear counterstain or is there any obvious reason not to do it?

I just wanted to let everyone know I tested the said protocol using both Alexa fluor 488 and Cy3-sulfonated. I only need 1ul of each azide though. They both came back with great results. I used it for cell cycle analysis on the Celligo Machine.

Hi,

Thanks everybody for the discussion it was very helpful for me !

Actually, I am working on a mouse reporter line with the fluorescent protein TdTomato. I injected EdU by i.p. (50mg/kg) and followed the protocol from Dr. Brumm for the revelation of EdU. I can see the positives cells for EdU but I cannot see anymore the cells positives for TdTomato and I have the same problem when I add (after the step of revelation) other antibodies.

Do you know if this process (for example the permeabilisation) could affect the stability of TdTomato protein ? Do you have any idea how I can solve this problem ? I'm gonna check if I can see TdTomato when I add an antibody anti-RFP.

Thanks !

Charlotte Baudouin there is a great change that un-chelated Cu quenches/damages fluorescent protein TdTomato. On potential solution is to use lower Cu concentration in combination with Cu chelator (for example THPTA) more reactive picolyl azides. Here is a link to a manual where you can find some additional compatibility chats (go to manual - Table 2) https://www.thermofisher.com/order/catalog/product/C10637

We also offer EdU Plus AF Imaging kits that are identical to Click-it Plus Alexa Fluor kits, here is a link to AF 488 kit:

https://clickchemistrytools.com/product/click-go-plus-edu-af-488-imaging-kit/

Cheers,

Andrei Polukhtin Thank you for your message ! As you recommend I've used a lower Cu concentration and it worked better. I also add the antibody anti-RFP to increase the signal for the TdTomato protein.

I am wondering if any of you have tried AF350 from Click Chemistry Tool and if you are able to use the protocol from Dr. Andrew Brumm's post with little modification.