4.4. Ex vivo DC Peptide Pulsing

Dendritic cells (DCs) were cultured and pulsed with peptide as previously described [24]. Haemopoietic stem cells were harvested from the bone marrow of the femur and tibia of BALB/c mice and seeded at 0.5 × 10⁶ cells /mL in RPMI (Gibco, Thermo Fisher, Waltham, MA, USA) supplemented with 10% FBS, 100 units/mL penicillin, 100 µg streptomycin, 2 mM L-Glutamine, 1 M Hepes (all Gibco, Thermo Fisher), and 0.1 mM 2 mercaptoethanol (Sigma Aldrich, St. Louis, MO, USA), plus 10 ng/mL GM-CSF (PeproTech, Cranbury, NJ, USA) and 5 ng/mL IL-4 (PeproTech, Cranbury, NJ, USA) for 6 days with half the media replaced on day 3. Cultured DCs were pulsed with 5 µg/mL SYIPSAEKI for 1 h and then washed with PBS and resuspended at 1 × 10⁷ cells/mL in PBS.