Day 0:

Activate CD4+ T cells O/N on plate-bound stimulation.

Day 1:

1. Pre-coat new plate with goat anti-hamster antibody (24 well for

2. Prepare tubes with RNA for transfection (1ul of 5uM prep per transfection)

• Set up Neon machine at this point.

3. Prepare plate- wash twice with PBS and add 1x anti-CD3 and -CD28 media with NO ANTIBIOTICS

1. Collect 3-4 tubes of blood in heparin vacutainers with green top (check expiration date!). You should get 1-2 million PBMC per ml of blood.

2. Use lymphoprep to isolate PBMCs.