Edu: 5-ethynyl-2’-deoxyuridine, a thymidine analogue, allows to detect DNA synthesis (S-phase) by using a reaction that makes incorporated Edu fluorescent, and thus, amenable to flow cytometry. The cell permeabilization required to expose incorporated intranuclear Edu uses saponins, the main permebilizer used in flow. We are able to detect Ki67 by saponin permeabilization. We want to determine if a cell population that epresses Ki67 is or has been in S phase.
Yes, it should work. We've done EdU stains with a number of antibody immunos in parallel. Invitrogen/Molecular Probes gives a combined protocol that works well. Order of steps matters because the Click-iT chemistry can destroy your fluorophore on the secondary antibody, so always do it the Click-iT chemistry step first, followed by IF.
I have done Edu staining on small number of cells say 0.1 million cells. Signal can be detected even with this small no.
I think it should be possible to detect both Edu and Ki-67 if you chose a right conjugate for Ki-67. I haven't used Edu but did use Ki-67.
Yes you should be able to detect both Ki-67 and Edu if you use different conjugated fluorochromes (ae: FITC and PE). You can gate on Ki-67 (ae FITC channel) and then from the gated population you look if there is Edu incorporation (ae PE channel).
Thanks for your answers. So, the reaction to activate EdU fluorescence does not interfere with subsequent intracellular Ki67 staining?
I have never assayed cell proliferation using both Ki-67 and Edu, but since Ki-67 is a nuclear protein and Edu will bind (incorporate) to DNA. There should not be any interference.
I have stained and detected both Ki67 and BrdU in the same cocktail in human epithelial cells. This was after fixing/permeabilisation and exposing the BrdU with DNAse I. Hope that helps.
Always important to use positive controls (E.g. Ki67 alone on proliferating cells, anti-EdU on cells not exposed to EdU) as I've found the intracellular binding is not as good with the wrong buffer or alternatively if the right buffer is used there can be a lot of non-specific binding.
Hope that helps.
We have done lots of BrdU stains with ki67 on same cells in same tubes using saponin .see my methods mol biol chapter for methods. Choose different colours for the 2 stains.
Yes, it should work. We've done EdU stains with a number of antibody immunos in parallel. Invitrogen/Molecular Probes gives a combined protocol that works well. Order of steps matters because the Click-iT chemistry can destroy your fluorophore on the secondary antibody, so always do it the Click-iT chemistry step first, followed by IF.
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