MLR and ELISPOT----- mix the T cells with the stimulator（cells），which one is more sensitive to the stimulator?
Dear Chen, The ELISPOT is probably more sensitive to smaller alloantigenic differences between stimulator and responder, but as the sensitivity increase so does the noise to non specific stimulation. Sometimes in the MLR and Elispot there is response to allogeneic and xenogeneic proteins so the tests should be carried out in serum from the responder
They kind of measure different things, so sensitivity is hard to compare
If you're measuring proliferation in your MLR by 3H-thymidine incorporation an IFNg ELISpot will be more sensitive. It might be worth using a CFSE-based proliferation assays are equally sensitive as ELISpot an allow you to analyze the responding cells (see attached papers).
Serum free media do work, but in our experience the cells function better in the presence of 5% serum. We routinely use pooled human male serum (see Optimisation of conditions... paper). This is fairly old now, but I hope still useful.
Good luck!
Stuart
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ELISPOT is measuring an early response, and is generally the most sensitive. MLR are measuring a more complex response to stimulation, but can also be very sensitive. It is possible that you will measure different types of cells with these two assays (terminally differentiated cells that produce cytokine might not proliferate, but rather die). If the response is so low, or you do not see it with both tests, you need to confirm with some other tests (cell death, surface markers) to be sure you fully understand the response you are measuring. We used human (but not autologous) serum for human tests and FBS for mouse tests. I would not do serum-free, first because it stresses the cells and second because serum contains proteins that bind to cell surface proteins and the plastic non-specifically and this coating seems to reduce non-specific responses due to spurious cell-cell interactions or "excess" adherence. If you are concerned about your particular situation (it might be sensitive to serum proteins) then you can try a serum substitute or modified serum. Again, when working with extremely low level responses, care needs to be taken to be certain that you are not measuring an artifact.
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