I stain NKT cells in the spleen by using NK1.1 and TCRβ. As was shown in the figure, I did detected NKT, but it was not very clear. I increased Ab concentration or PMT voltage, but it did not work. I would appreciate it if anyone could give me some suggestions.
The separation looks clear enough and comparable to published data. Increasing the antibody concentration may lead to nonspecific binding and increasing the voltage might not be helpful. You can try using antibodies with different fluorophores with stronger intensity which might give you better resolution. I hope this information helps. Good luck
Like Ramachandramouli suggested, the separation is good. It could be that compared to NK/T cells, your NKT cells have lower expression of NK1.1/TCRb, leading to a population with overall lower signal.
Increasing the antibody concentration would not give you better separation between the positive and negative populations. Most likely, both populations would shift higher on your axes. Increasing the voltage would do the same. And your NK1.1 is already conjugated to one of the most intense fluorophores.
If you want better separation, try alternate antibody clones from different vendors. Most of them have representative FACS plots on their product sheets. Pick the ones which show a good separation between the two populations. I have tested up to 6 different clones for some of the more difficult markers, before picking the one that gave the best separation.
That actually looks like a pretty clear separation. If you are trying to increase the intensity, you should probably try a different fluorophore.
Increasing Ab concentration will not do anything, since we almost always use saturation levels of Abs compared to marker.
The only other thing I can think of is to double check your compensation settings. You may be able to move the population higher. But this of course will mean that you will need to rerun your samples under the new protocol.
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