OT-1 T cell Priming Protocol

DsRed fluorescent protein-expressing OT–1 mice (C57BL/6–TgTcra/Tcrb homozygous, 1100 Mjb/J) were a generous gift from Professor Mei Wu (Wellman Center for Photomedicine, Massachusetts General Hospital and Harvard Medical School, Boston). Mice 10 weeks of age or older were euthanized by CO₂ asphyxiation following an approved IACUC protocol. Spleens were collected aseptically immediately after euthanasia and T cells were primed following a published protocol (39 (link)) with minor modifications. Briefly, red blood cells in the splenic suspension were depleted by incubating with ammonium–chloride–potassium lysing buffer (Gibco, A1049201) for 4 minutes. Three days before addition to 3D cultures, T cell blasts were generated in 6–well plates (Corning, 353046) by inoculating 0.75 μg/mL of SIINFEKL peptide (ovalbumin sequence 257–264, New England Peptide, BP10–915) into 1×10⁶ splenocytes/mL for 3 days in the presence of 10 U/ml recombinant murine interleukin–2 (IL–2) cytokine (Peprotech Inc., 212–12). This protocol yields a >90% pure solution of CD8+ T cells (39 (link), 40 (link)) and was not purified further. Cells were washed twice in RPMI 1640 media before addition to 3D cultures. In all cases, T cells were maintained in SuppRPMI medium further supplemented with 50 μM β–mercaptoethanol (Sigma, M3148) and 10 U/mL recombinant murine IL–2 (T cell RPMI).