Interferon-gamma (IFN-γ) enzyme-linked immunospot (ELISpot) assay
SEPARATION
IFN-γ ELISPOT kit (Shenzhen Dakwei Biotechnology Co., Ltd, Anshan, China) was used to perform the ELISpot assay for detection of IFN-γ in neoantigen-specific T cells. Peripheral blood mononuclear cells (PBMC) separation was performed with 10 mL of peripheral blood, while dendritic cells (DC) and T cells were separated by a 6-well plate culture. Thereafter, DC and T cells were mixed in a ratio of 10:1 and aliquoted into 96-well plates for co-stimulation culture; 200 µL Rosewell Park Memorial Institute medium (RPMI 1640) containing 100 U/mL IL-2, 10% fetal bovine serum, and 4 µg polypeptide was added to each well. The co-stimulated cells were then transferred to the Elispot plate. While 1/1000 CD3 and phosphate-buffered saline (PBS) solution were applied to the positive control and negative control wells, 4 µg polypeptide was added to the polypeptide well. The cells were grown in a CO