Live dead violet viability kit

Directly conjugated antibodies against murine CD3e (17A2), CD4 (RM4-5), CD8b (YTS156.7.7), IFN-γ (XMG1.2), TNF-α (MP6-XT22), and IL-2 (JES6-5H4) were purchased from BioLegend. A Live/Dead Violet viability kit (Invitrogen) was used to exclude dead cells from analysis. To determine intracellular cytokine production, 2 million splenocytes from vaccinated mice were cultured with peptides (5 μg/mL) derived from corresponding mutated neoantigen, protein transport inhibitor (eBioscience), and CD107a antibody (1D4B, BioLegend) for 5 h. Surface staining followed by intracellular cytokine staining was done to determine cytokine production. Cells were permeabilized using the eBioscience FoxP3 staining kit as per the manufacturer’s instructions. Data were acquired on a BD FACSymphony (BD Biosciences) and analyzed using FlowJo. The neoantigen peptides consisted of 15-mer peptides overlapping by 9 aa. The peptides were designed to cover the entire 33-mer used for vaccination. Mice were vaccinated three times at 2-week intervals and euthanized 1 week after the last vaccination. Spleens were harvested, and splenocyte suspensions were obtained using a Stomacher 80 Biomaster (Thomas Scientific), followed by red blood cell lysis (Thermo Fisher Scientific).