I am going to use Click-iT Edu reagent and protocol to detect cell proliferation by flow cytometry, on cells staining for extracellular and intracellular proteins. The reagents are compatible with R-PE and R-PE conjugates (i. e. PE-Cy7), if the antibodies conjugated with them are added AFTER the Click reaction.
I am using PE and conjugates for staining of membrane extracellular proteins (CCR7, CXCR3, CD45RO), and I should stain for them after permeabilization, intracellular Mab addition and EdU develop reaction. I would appreciate any comment.
You can stain for these markers after permeabilisation. However, permeabilising will allow your antibodies access to these markers inside the cell (e.g. in the cytoplasm). If you are only interested in whether these cells are positive or negative for these markers then this is not a problem.
I guess for me if I had stained my cells after fixing and perming then I would look at a marker like CCR7 and go '"reat I have CCR7+ staining... but how much of that is actually on the cell surface? Can those CCR7+ cells actually home to the lymph node in reality?"
Thanks, Anthony and Rachel. I'll give it a go, and might validate it with pre-perm surface staining.
Depends on whether your marker is only at the cell surface or also inside cells.
Why do not you stain before on the surface and then inside?
To see a marker on proliferating cells it is better to use CFSE or violet staining (see BD) and then stain cells at the cell surface.
In agreement with Alessandro, I also wonder why you don't perform the surface staining 1st then proceed onto permeablizing and fixing for intracellular staining. I haven't performed cell proliferation assessment by flow, so maybe that is the reason you have to do it in that order.
In any event, another consideration is that if you have to permeabilize and fix 1st, will the surface epitope is still be recognized by the surface staining antibody? Some antibodies work with fixed tissue others do not.
Good luck.
It really depends if the epitopes which your mAbs are against are damaged/changed during fixation. It would be wise to test this before doing the entire experiment. Stain prior to using the fix/perm step and also have a set of stains afterwards. It would probably be a good idea to do an Ab titration under these conditions, because the results are likely to vary depending on the fixation state.
Whenever I do dual staining of surface markers and intracellular markers I have seen staining of surface markers before the intracellular markers give better results. what I do is add brefeldin to my culture, incubate for 4 hours proceed with surface markers staining and then add perm buffer and proceed with the intracellular staining. pemeabilization do change the surface epitopes sometimes. But if the given cell proliferation assay needs intracellular staining before surface staining then it is fine I guess.
I agree with Paula on this. In my experience (anecdotal, though it is), CD8 (human and monkey) definitely does not survive the permeabilization reagent (using BD perm 2), and I got different results with CD4 in humans than in monkeys (different epitopes were targeted), and other markers were very difficult to interpret, due to excessive non-specific staining in those channels. Once you permeabilize, there is a the wealth of intracellular proteins that will non-specifically pick up any antibody stain. I think you need to do a small, well-controlled pilot experiment with your markers of interest, using the same or similar cells as you will use in your actual experiment, as that is really the only way to know whether they will survive the permeabilization reagent.
In general, the two-step process is used for two reasons: 1) because the perm reagents tend to mess up surface epitopes which are stabilized once they are stained with conjugated antibody, and 2) because there is so much more opportunity for background/non-specific staining in a permeabilized cell. It is VERY important that you use isotype controls in your permeabilized cells (at least once) to determine how much non-specific baseline staining you have - even in assays designed for intracellular use - in order to prevent reporting false-positive, possibly non-repeatable results.
Just out of curiosity, have you explored the possibility of using an alternate proliferation assay that does not require permeabilization, such as CFSE? This would enable you to to avoid your permeabilization issue.
Jessica, thank you very much for your useful answer. I'll run a pilot, and if it does not work I will certainly use CFSE. Fluorescein and derivates are not harmed by the click-iT reaction. Thanks for the input.
You won't need CFSE if you're using a Brdu type assay, you could do both but they're informative for different reasons. As for CcR7, the receptor is continuously being endocytosed, so it is optimal to stain live cells at 37deg. I guess you could try and see if perm'd cells will also stain well. Due to that continuous cycling, I would not worry about "true, surface ccr7".
In general, formaldehyde fixation causes changes that will disturb the epitopes on the cell surface, so up your surface stain concentration 2X of normal to get the same brightness.
look up Mario Roederer's paper on chemokine receptor staining (J immunological methods 2003 I think). I do my surface stainings before fix/perm but I realize this is not as possible due fluorochrome quenching by the click reaction.
If you need a short read time (
I do have the same konfusion i am staining only surface markers so i think i dont need to permeabilize the cells.
Komal,
In oyour case, you absolutely don´t need to worry, except if you want to do the click reaction for EdU staining, and are using PE orPE-derived fluorochromes.
thank u
i am using PE antiCD135 for surface staining. is it ok that stain the cells directly with out permeablization
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