Sodium pyruvate

DCs were generated from murine bone marrow cells, as described by Garrigan et al^{23 (link)} with minor modifications. Briefly, bone marrow was flushed from the long bones of C57BL/6 mice. A single cell suspension was cultured in RPMI 1640 (CellGro, USA) supplemented with 10% heat-inactivated FBS (Life Technologies, USA), 2 mM L-glutamine (Gibco, USA), 100 U/mL penicillin (Gibco, USA), 100 µg/mL streptomycin (Gibco, USA), 10 mM HEPES pH 7.4 (Gibco, USA), 0.5 mM sodium pyruvate (Cellgro, USA), 0.5% MEM non-essential amino acids (Cellgro, USA), 0.1 mg/mL Normocin (InVivogen, USA), along with 20 ng/mL GM-CSF (Peprotech) and 10 ng/mL IL-4 (Peprotech, USA) for 4-DCs or 20 ng/mL GM-CSF and 250 U/mL IFNα (AbD Serotec, USA) for α-DCs. On day 2, the medium was replaced with fresh medium containing the respective cytokines. On day 4, 100 µL of the indicated tumor lysate preparations were added to 10 mL culture (~1 tumor cell equivalent per DC) and incubated overnight at 37°C and 5% CO₂. Unpulsed DCs were also cultured as negative controls. On day 5, the medium was removed and fresh medium containing 100 ng/mL LPS (Sigma-Aldrich, Germany) and 100 ng/mL IFNγ (Peprotech, USA) was added. DCs were then cultured overnight at 37°C and 5% CO₂.