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Plat-A cells (8×105 cells/well) were seeded 24h before transfection in 6-well plates in 2 mL/well of infection medium (Plat-A medium without antibiotics). 20-24h later, as soon as the cells had reached 70% confluence, 500μL/well of a combination containing plasmids and Lipofectamine had been added. The mixtures contained 3 μg/well of transgene carrying plasmid, 2 μg/well of pMD2.G and 10 μL/well of Lipofectamine 2000, and were prepared in OPTIMEM medium according to the manufacturer´s protocol.

As proven in Figure 5D, these T cells particularly acknowledged neoAg-expressing tumor cells. NeoAg-specific responses had been evaluated at day 21 of co-culture through the use of a human IFN-γ ELISPOT set (BD-Biosciences) following manufacturer’s instructions. Expanded CD8 T cells (5 x 104/well) had been stimulated with peptides (10 µg/mL) and DC (104/well) for twenty-four h.

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