Ova257 264 siinfekl

Ten days after tumor injection, spleens were harvested on euthanized mice. Single cell suspensions of splenocytes were obtained using a 70 μm cell strainer. Cells were washed in PBS before the red blood cells were lysed in ACK buffer (Lonza, Basel, Switzerland) for 4 min and blocked with complete media (IMDM +10% FBS +1% P/S). Cells were centrifuged, resuspended in complete media, and 0.5 million were plated in in 96 U-bottom plate. OVA_257-264 (SIINFEKL; GenScript) and OVA_323-339 (ISQAVHAAHAEINEAGR; GenScript) were added to the splenocytes at a final concentration of 1 μg/mL to restimulate CD8⁺ and CD4⁺ T cells, respectively. Unstimulated controls were tested using complete media without peptide, and positive controls were tested using ionomycin (1 μg/mL) + PMA (50 ng/mL). After 4 days in culture, the cell supernatant was collected and the amount of ΙFNγ secreted was quantified using mouse IFNγ quantikine ELISA kit (R&D systems, Minneapolis, MN, USA) according to the manufacturer’s instructions. Data represent the concentration of IFNγ secreted in restimulated culture supernatants subtracted with the amount detected in unstimulated supernatants.