I have been activating T cells with anti-CD3 (either soluble or plate bound) for many years.
Currently, I am establishing a protocol whereby at the time of coating (or subsequently) I plate bind my molecule of interest to the plate (1-step and 2-step coating, respectively).
My results show that my compound decreases anti-CD3-mediated T cell activation.
However, I recieved comments that I see less activation because my coumpound "displaces" the bound anti-CD3, and therefore my results are not decreased activation via my compund; but in-fact are an artifact, and the decrease is due to decreased levels of anti CD3 in the well.
Does any one know how to over come this displacement?
soluble anti-CD3 is not an option for me currently (Technical reasons).
Hi,
What are your controls? You can add another stimulation control by coating the plate with CD3 mAb + isotype control Ab of same concentration to that of your molecule of interest. If you consider this possibility then I would also include additional control with stimulating the cells only with isotype control.
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