Primary bone marrow-derived DCs were flushed from the femurs and tibiae of mice in sterile conditions, and incubated in RPMI 1640 medium at 37 °C in 5% CO2. On Day 3, non-adherent cells were discarded. The cells were further cultured for 4 days in fresh medium containing GM-CSF (10 ng/mL) and IL-4 (10 ng/mL). On Day 7, DCs were incubated in the presence of freeze-thawed B16 tumor lysates. B16 melanoma cells were lysed by rapid freezing (liquid nitrogen) and thawing at 37 °C in physiological saline three times. After 24 h, LPS (1 μg/mL) was added into the culture for DC-maturation for another three days. The mature B16 melanoma cells lysate-pulsed DCs were harvested, which was determined using- flow cytometer (Beckman Coulter, Brea, CA, USA) after incubating with CD80-FITC.