Figure 3.

ELISpot screening to identify T cells that recognize tumor-specific peptides in untreated and LIT-treated B16-F10 tumor-bearing animals. (A) Schematic of performing ELISpot for peptide screening. C57BL/6 male and female mice of 6–8 weeks old were injected with 1e5 B16-F10 tumor cells. Once the tumors reached 0.5 cm³, the tumors were treated with LIT or left untreated. Eight days after treatment splenocytes were isolated and used for ELISpot. (B) Approximately 5e5 total splenocytes were used for the ELISpot with eight selected peptides (~1 µM for each peptide). ELISpots were incubated for ~36 hours prior to developing the ELISpot. Spots were counted using the default settings on the ELISpot reader. Assays were performed in duplicate. The average number of spots in the negative control was then subtracted from the average number of spots in the peptide incubated wells to get the final spot count. LIT, laser immunotherapy; SQ, subcutaneous.