Optimization of PBMC preparation for use in the tumor IFN-γ ELISPOT assay. Cryoperserved PBMC were thawed and rested in culture medium either overnight (O/N) or for 2 h and assessed for optimal reactivity to tumor cells. Pre- (white columns) and postvaccine (grey columns) PBMC (1 × 105 cells/well) were co-cultured with sCD40Lt activated autologous tumor cells (2 × 105 cells/well) for 48 h in the ELISPOT assay. Significantly (p < 0.05) more IFN-γ production by responding T cells was observed in post- compared to prevaccine PBMC when PBMC were rested overnight. Data is presented as average number of spots per well ± SD and is representative of four experiments with similar results.