Mouse T Cell Isolation and Activation

B6.SJL-Ptrc^a Pepc^b/BoyJ(CD45.1 mice) were euthanized and spleens were collected. After tissue dissection and red blood cell lysis, primary mouse T cells were purified using the mouse Pan T cell Isolation Kit (Miltenyi Biotec; 130-095-130). Purified T cells were cultured in RPMI-1640 (Invitrogen; 11-875-085) supplemented with 10% FBS (Corning; 35–010-CV), 10 mM HEPES (Thermo Scientific; 15630080), 2 mM L-glutamine (Thermo Scientific; 25030164), MEM non-essential amino acids 1x (Thermo Scientific; 11140076), 55 μM β-mercaptoethanol (Thermo Scientific; 21985023), 1 mM sodium pyruvate (Thermo Scientific; 11360070), 100 IU ml⁻¹ recombinant human IL-2 (Proleukin; Novartis) and mouse anti-CD3/28 Dynabeads (Gibco; 11452D) at a bead:cell ratio of 1:2. T cells were spinoculated with retroviral supernatant collected from Phoenix-ECO cells 24 h after initial T cell activation as described^{79 (link),80 (link)} and used for functional analysis 3–4 days later.