OKT3 is used as an immunosuppressive antibody which targets the CD3 molecule that is present exclusively on T cells and depletes them from the circulation. You may try to stimulate CD4 T cell by plate bounded anti-CD3 ε and soluble anti-CD28 antibodies. The antibodies we used are anti-mouse CD3 ε (clone: 145-2C11) and anti-mouse CD28 (clone: 37.51). The brief protocol as follow: 1. Coat culture plates with anti-CD3 antibodies (at concentration of 1-2 µg/ml) overnight at 4°C. 2. Wash plates to remove unbound antibodies by PBS. 3. Add anti-CD28 antibodies to the plates (at concentration of 1 µg/ml) just before adding the cells. 4. Seed cells onto the antibody-coated plates at 37°C in a 5% CO2 incubator. The incubation time may vary (usually 24-72 hours) depending on the experimental setup. 5. After a certain period of incubation, add Brefeldin A or Monensin to the culture to inhibit the secretion of CD107a, trapping it inside the cells and incubate for an additional 4-6 hours. 6. Harvest the cells and wash them in PBS. Stain cell with a fluorochrome-conjugated anti-CD4 antibody and then fix and permeabilize cells with appropriate buffers. 7. Stain permeabilized cells with a fluorochrome-conjugated anti-CD107a antibody and analyze the stained cells using flow cytometry to detect CD107a expression in CD4 T cell population.

Be sure to use antibody coated plates or beads for stimulation. Soluble antibodies are not efficient for stimulation (other than for whole blood). Staphylococcal enterotoxin b is also an option, but only a small percent of T cells (in PBMCs) is activated.