I plate 96-well plates with anti-CD3 O/N at 4*C. I use these plates for T cell proliferation assay with MDSCs. I think my assay didn't work because I didn't do adequate washes and might have left too much of anti-CD3 behind in the wells which resulted in non-specific stimulation of T cells even in the presence of MDSCs.
Hi Jaspreet
It's a possibility. If you consider Dynabeads (I know it's a different assay) they say that if you use excess beeds may inhibit expansion. How much anti CD3 are you using? How did you wash your cells?
It appears that your T cells are proliferating but MDSCs are not able to execute their suppressive function.
At what ratios have you added MDSCs to your T cells and what is the source of MDSCs you isolated. How pure are they.
What else have you used for the stimulation of T cells and how much of cells have proliferated in the absence of MDSCs.
Have you tried to cover your plates and incubate for 30 min at 37 degrees and then wash? Is possible than an excess of stimulation could induce anergy or exhaustion in T cells, but maybe your problem is the adherence of the antibody.
