Mouse ifn γ elispot kit

ELISpot analysis was done using a mouse IFN-γ ELISpot kit (Abcam, Waltham, MA USA). Murine lymphocytes obtained from inguinal lymph nodes (1 × 10⁵ cells/well) were loaded into a well of 96 well plates. Lymphocytes were stimulated with each peptide (1 µg/well) for 24 h at 37 °C, 5% CO₂. Peptide sequences are summarized in PHArmaceutics-14-00093-t001">Table 1. Lymphocytes treated with phytohemagglutinin (1 μg, PHA; PanEco, Moscow, Russia) were used as positive control. Cells without treatment were used as the negative control. Each peptide was analyzed in duplicate.

The relative numbers of splenic PyCS-specific, IFN-γ-secreting CD8⁺ T cells of AdPyCS- or RAS-immunized mice were determined by an ELISpot assay, using a mouse IFN-γ ELISpot kit (Abcam, Cambridge, MA) and a synthetic 9-mer peptide, SYVPSAEQI (Peptide 2.0 Inc., Chantilly, VA) corresponding to the immunodominant CD8⁺ T cell epitope within PyCS, as previously described (32 (link)). Briefly, after the collection of splenocytes from mice 12 days after AdPyCS or RAS immunization, 5 × 10⁵ splenocytes were placed on each well of the 96-well ELISpot plates pre-coated with IFN-γ antibody and incubated with the peptide at 5 μg/mL for 24 h at 37°C in a CO₂ incubator. After the ELISpot plates were washed, they were incubated with biotinylated anti-mouse IFN-γ antibody for 2–3 h at RT, followed by incubation with avidin-conjugated with horseradish peroxidase for 45 min at RT in the dark. Finally, the spots were developed after the addition of the ELISpot substrate (Abcam). To identify the number of IFN-γ-secreting CD8⁺ T cells in each well, the mean number of spots (for duplicates) counted in the wells incubated with splenocytes in the presence of the peptide was subtracted by the mean number of spots (for duplicates) counted in the wells that were incubated with splenocytes only.

The relative numbers of PyCS-specific, IFN-γ-secreting CD8⁺ T cells among whole splenocytes isolated from the spleens of immunized mice were determined by an ELISpot assay, using a mouse IFN-γ ELISpot kit (Abcam, Cambridge, MA, USA) and a synthetic 9-mer peptide, SYVPSAEQI (purchased from Biosynthesis Inc.; Lewisville, TX, USA) corresponding to the immunodominant CD8⁺ T-cell epitope within the PyCS antigen, as previously described (32 (link)–34 (link)) with some modifications. Briefly, splenocytes were prepared by lysing red blood cells from a single cell suspension obtained from a spleen that was collected from mice 12 days after immunization, and 5 × 10⁵ splenocytes/well were incubated with 5 μg/mL of the peptide for 24 h at 37°C on the ELISpot plate pre-coated with an IFN-γ antibody, as previously described (32 (link)–34 (link)). Subsequently, the ELISpot plate was incubated with biotinylated anti-mouse IFN-γ antibody, followed by incubation with avidin-conjugated with horseradish peroxidase. Finally, the spots were developed after adding ELISpot substrate (Abcam). To identify the number of IFN-γ-secreting CD8⁺ T cells in each well, the mean number of spots (for duplicates) counted in the wells incubated with splenocytes in the presence of the peptide was subtracted by the mean number of spots (for duplicates) counted in the wells that were incubated with splenocytes only.