The SB525334 nmr number of induced spots was dose-dependent and increased in the presence of higher number of target cells up to 2 × 104. Peak concentration corresponded to 2 × 104 target cells. Higher concentrations did not lead to a significant increase in spots (P = 0.14). Figure 3 Panel A – LysiSpot assay. LysiSpot assay results, expressed as net number of spots per well (spots from wells containing only target cells were subtracted), from four different experiments (mean ± SD). Increasing numbers of target cells were plated in short term cultures with

effector cells (2 × 105/well PBMC). Spots were the imprint of β-gal, released by the transfected DHD-K12 target cells after lysis. Cytotoxic activity of PBMC from DHD-K12-inoculated rats or control rats are represented by dark and light grey respectively. Panel B – LDH-Cytotoxicity assay. Cytotoxic activity expressed as percent

of specific lysis (mean ± SD) of DHD-K12 target cells from PBMC of intact (control) or DHD-K12-inoculated rats (Primed) evaluated by Promega CytoTox 96 kit. NVP-HSP990 cost Concentration ratio of effector and target cells was 10:1 (light grey), 5:1 (dark grey), 2.5:1 (white), 1.25:1 (black) and corresponding Thiazovivin respectively to 2 × 104,1 × 104, 5 × 103, 2.5 × 103 of DHD-K12 target cells. To further demonstrate the in vitro specific cytotoxicity of PBMC from intact or DHD-K12-inoculated rats against DHD-K12 cell line we utilized a colorimetric assay (CytoTox 96 kit Promega) that quantitatively measures the release of lactate dehydrogenase 6-phosphogluconolactonase (LDH) from killed tumor cells. In Figure 3B the results, expressed as percent of specific lysis confirm, at comparable effector: target ratio used in Lysispot, the specific cytotoxic activity against DHD-K12 tumor cell line. Cytotoxicity and IFN-γ secretion evaluated by the dual-colour LysiSpot assay The dual-colour assay allowed to determine both the induction of cytotoxic effects in association with the production of IFN-γ in response to the specific recognition of the tumor cells. DHD-K12 β-gal transfected cells (2 × 104) were cultured with 2 × 105 PBMC from control or tumor harbouring rats. Trough the combined

analysis of the spots of different colours, a differential counts of the number of lysed cells (pure red spots), the number of PBMC secreting IFN-γ (pure blue spots) and the number of cells that simultaneously secreted IFN-γ and lysed the targets (violet spots combining both colours) was allowed. The histograms depicted in Figure 4, represent the results of three different experiments and show that IFN-γ secretion and cytotoxicity are distinct CTLs functions that can be independently regulated. Therefore, in our experimental conditions, 55% of the overall immune activated cells developed a full lytic activity and a large portion of these cells (65%) also released IFN-γ. The remaining 45% produced IFN-γ but were not cytotoxic. Figure 4 Dual-colour LysiSpot assay.