BMDC-T Cell Co-culture

Following a standard protocol,32 BMDCs were generated ex vivo from isolated bone marrow cells of BALB/c mice in the presence of murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-4. BMDCs were seeded into 96-well U-bottom plate at 2 × 10⁴ cells per well in the presence of 10 μg/mL TWIST1 or OVA peptide and incubated at 37°C for 1 h, followed by exposure to 1, 10, or 100 μg/mL anti-CTLA-4 antibody (clone 9D9) at 37°C for 1 h. Then, CFSE-labeled CD3⁺ T cells (1 × 10⁵) were co-cultured with antigen-pulsed BMDCs for 6 days before analysis by flow cytometry. Half of the culture medium was replaced every 2 days, and IL-2 (10 ng/mL) was added into the co-culture at day 4.