Primary naïve CD8+ T lymphocytes isolation and stimulation

Spleen and lymph nodes were collected in GentleMACS C-tubes (cat # 130-096-334) containing 5 mL complete medium (RPMI 1640 with Glutamax- Gibco cat # 61870–036, 10% heat inactivate FBS- hyClone cat # SH3008803HI, 1mM sodium pyruvate -Gibco cat # 1136–070, 55uM b-mercaptoethanol- Gibco cat # 21985–023, 10mM HEPES pH 7.2). Tissues were homogenized with a GentleMACS OCTO dissociator. CD8+ T lymphocyte isolation was performed with Dynabeads untouched mouse CD8 cells kit (cat # 11417D, Invitrogen), according to the manufacturer’s protocol. Isolated cells were counted and resuspended at 2x10⁶/mL in complete medium. 4x10⁶ cells were plated in each well of 6 well plates pre-coated with anti-CD3e antibody (BD Pharmingen purified NA/LE hamster anti-mouse CD3e clone 145-2C11, cat # 553057) at 10ug/mL, or non-coated plates for control, and incubated at 37°C for 1, 2, 3, 4, 24 hours. Non-stimulated (Naïve) samples were kept on ice until fixation.