Hopefully, you have a good MHC II tetramer. In my experience, getting an MHC II tetramer with good, reproducible binding has been difficult.

I have never tried this particular approach, but I do not see why they would interfere.

However, I would recommend a trial staining with MHC I tetramer alone, MHC II tetramer alone, and both MHC I and MHC II tetramers. If you are working with splenocytes you should easily get enough cells to set up all three staining conditions.

Then compare the results of single- and two-tetramer staining; if you see a drop off in the percentage of either tetramer when the other is present, you have your answer.

Hopefully, you have a good MHC II tetramer. In my experience, getting an MHC II tetramer with good, reproducible binding has been difficult.

I have never tried this particular approach, but I do not see why they would interfere.

However, I would recommend a trial staining with MHC I tetramer alone, MHC II tetramer alone, and both MHC I and MHC II tetramers. If you are working with splenocytes you should easily get enough cells to set up all three staining conditions.

Then compare the results of single- and two-tetramer staining; if you see a drop off in the percentage of either tetramer when the other is present, you have your answer.

As far as I remember MHC I and II peptide pocket grooves have specific conformations to accept peptides of defined length (9 mers for class I and about 20 mers for class II). So, I think that if your tetramers are specifically designed to fit with MHCI or MHC II respectively, incubating splenocytes with both tetramers at the same time should not affected their own binding. However, Matthew gave the good method to experimentally answer this question.

hope it helps

Thanks for the replies! I was going to do the same thing that Matthew suggested but didn't want to do it if someone had already done it before.

I can't see any reason why they would interfere. The MHCII tetramers should bind to your CD4+ T cells, while the MHC I tetramers should bind to CD8+ T cells and possibly also to your NK cells. You should definitely also stain with each one alone, and you should also probably co-stain with CD3, CD4, CD8, and NK1.1 so that you know what cells they are binding to.

One concern would be the interference of biotin and streptavidin from the two tetramers. Tetramers are generally made by binding 4 biotinylated monomers to 1 molecule of fluorophore-conjugated streptavidin. If any of the streptavidin sites are free, there is the possibility that one they might bind each other. I don't know how likely this is going to occur though.

The main value of tetramers is that the MHC:TCR binding is highly-specific, so direct cross-reactivity/cross-interference should be minimal (as stated by many of the above). Sue Tsai makes a good point about possible binding to streptavidin. However, if the tetramers bind to one another via streptavidin, they should not bind to your cells. Unless this occurs at high frequency, it should not interfere with the signal you get by flow cytometry (i.e., should not see cells double-positive for MHC I and MHC II).

You should note that the best protocls for class I and class II tetramer binding differ-generally class I tetramers bind better, ut class II seem to need more time- maybe to endocytose the TCR:tetramer complex to be stable staining, so usually done at 37 for 60 min while class I binding is faster and usually done at RT or 4.

No matter what the contyrols noted aboe by Matthew should be done for every tetramer pair. Also remember that the freqeuncy of CD4/Tet+ cells is likely to be a lotl ower than CD8/tet+ cells.