i do not really know BrdU labelling, however, it demands a harsh handling of samples to retrieve the epitop. It is not really compatible with IF (double), i guess. If you find method for BrdU with double labelling against an additional antigen, you can use that technique (click reaction first, than BrdU labeling, i guess).

Attila Lehotzky Thank you for your answer! Yes, I was thinking about using the Thermo Fisher protocol (C10420) and refer to the manual section where it describes the double pulsing procedure.

Dear Andrea, You do not say why you want to pulse cells with BrdU and EdU - they label the same part of the synthesized DNA - is this a competitive binding assay? Also you need to used 2N HCl to partially unwind the DNA to label BrdU and Click chemistry to label EdU - so doing these two things together would probably not work as the HCl would maybe stop the EdU from being tagged by the Click chemistry. As Attila said the point of EdU labelling as opposed to BrdU it is easier to then label for an additional antigen but not brdU as this requires the HCl - after all that's what EdU labelling with Click chemistry allows you to do. Also these are all standard methods commonly used in flow cytometry which can be used for fluorescence microscopy too, just do a search on the Cytometry A Journal website and you will find lots of publications Regards Gary

@Gary Warnes thanks for the answer! I am aware of the limitations of BrdU. I would like to do a first EdU pulse, follow that with a second BrdU pulse and then stain with Click chemistry to detect EdU and treat to unwind DNA and detect BrdU. I am wondering if at this point the double stained sample can be analysed by facs. In theory I would distinguish EdU+/BrdU+ cells from double negative or single positive cells (providing BrdU and EdU are labeled with different color).

I still do not understand why you want to pulse with both EdU and BrdU as they measure the same thing? Also to label the BrdU you need to use 2N HCl which is incompatible with any fluorescence marker! So this dual labelling cannot be done for flow cytometry or any fluorescecne based instrumentation, Regards Gary

Andrea Tavosanis Yes, that protocol is a good one and can be used with flow, as we show in Figure 1 of the manual you reference (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Click_iT_EdU_Flow_Cytometry_Assay_Kits_100_tests_PI.pdf).

One thing to keep in mind is that you will need a clone of anti-BrdU antibody that does not recognize EdU. Thermo Fisher sells the MoBu-1 clone, which you can use.

Gary Warnes Both EdU and BrdU do incorporate the same way, incorporating into DNA during cellular proliferation. But in some conditions you want two different windows of time on the same cells. If so, then you'd need different reporting for those different times. BrdU is detected with antibody of one color, while EdU is detected with a Click reaction using a different color.

The HCl wash for BrdU will kill GFP and some other fluorescent proteins, but it won't affect the ability of other dyes that are introduced later, such as secondary conjugates or azide derivatives (for Click reaction). But even GFP can be recaptured using an anti-GFP antibody if needed.

Jason A Kilgore Thank you for the answer and for making my point more clear! I will work towards trying this kind of assay for sure