I just started performing 3H thymidine incorporation assays. After isolating lymophocytes from a mouse spleen using Lymphoprep, I activated 2x105 cells/well in a 96 well plate using 5-10 ug/mL of plate bound anti-CD3 (145-2C11 clone) +/- soluble anti-CD28 (37.51 clone) at 2 ug/mL. I noticed a huge drop in proliferation in the wells where I added anti-CD3 AND anti-CD28 compared to the wells that had only anti-CD3. I thought anti-CD28 was supposed to increase activation. Any thoughts?