Ifn γ elispot set

Responses induced by vaccination were measured using IFN-γ ELISPOT Set (BD-Biosciences). Splenocytes (5 × 10⁵/well) were stimulated with OVA(257-264), OVA (323-339), AH1-A5 or ELA (10 μM) for 24 hours at 37 °C and the number of spot-forming cells was counted using an ImmunoSpot automated counter. Non-stimulated cells were used as controls.

Responses induced by vaccination were measured by using an IFN-γ ELISPOT Set (BD-Biosciences, Franklin Lakes, NJ, USA). Briefly, splenocytes (8 × 10⁵ cells/well) were cultured in ELISPOT plates and stimulated with OVA or GPC3 CD4/CD8 epitope peptides (10 μg/mL), OVA protein (10 μg/mL), GPC3 peptide pools (1 μg/mL of each peptide) or irradiated tumor cells (8 × 10⁴ cells/well). One day later plates were washed, developed and spot-forming cells were counted automatically.

Most T cell responses were evaluated by using an IFN-γ ELISPOT Set from BD-Biosciences as described (30 (link)). In brief, spleens were removed, homogenized, erythrocytes were lysed and cells (8×10⁵/well) were stimulated with different peptide concentrations (10-0.01 μM). To analyze recognition of neoantigen-presenting tumor cells, irradiated (200 Gy) MC703 cells (8×10⁴ cells/well), transduced with retroviruses encoding neoantigens or with control vector, were cocultured with splenocytes (8×10⁵/well) obtained from immunized mice. In all cases, 24 h later ELISPOT plates were developed and spot-forming cells were counted with an ImmunoSpot automated counter using Immunospot Image Acquisition 4.5 and Immunospot 3 software.
In some cases, T cell activation was determined by flow cytometry. Splenocytes were stimulated for 4 hours with peptides (10 μM) in the presence of GolgiStop and GolgiPlug (BD-Biosciences) and antiCD107a-FITC (BD-Pharmingen). Next, they were stained with anti-CD3ϵ-Percp-Cy5, CD4-FITC and CD8-BV421 (BioLegend). Then, cells were fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation/Permeablization Kit and intracellularly stained with IFNγ-PE and TNFα-BV510 antibodies (BioLegend). Dead cells were excluded from the analysis using Maleimide (PromoKine). Samples were acquired with a Cytoflex cytometer (Beckman Coulter) and data analyzed using FlowJo software.