Murine ifn γ elispot kit

Isolated splenocytes were stimulated at 4 × 10⁵ cells per well (Multiscreen-IP PVDF Filter plates, Milipore) for 20 h with 5 µg/ml of peptides (Eurogentec). The NP peptide (147–155) is the cross-reactive H-2K^d-restricted epitope derived from the influenza A H1N1 PR8 virus comprising the amino acid sequence TYQRTRALV and is sequence identical between influenza A H1N1 PR8 and influenza A/NL/18/94 H3N2. This peptide was shown to constitute the immunodominant epitope of NP in BALB/c mice [25 (link)].
The HA peptide (518–526) is a H-2K^d-restricted epitope derived from the influenza A H1N1 PR8 virus comprising the amino acid sequence IYSTVASSL. For intra-assay negative and positive controls, medium (Mock) or Dynabeads Mouse T-Activator CD3/CD28 beads (Thermofisher, data not shown) were used, respectively. IFN-γ detection was performed using the murine IFN-γ ELISPOT kit from Diaclone. Spot forming cells (SFC) were counted using an ELISPOT reader (Autoimmun Diagnostika GmbH, Germany) and are shown after background (mock conditions) subtraction.

In all experiments a murine IFN-γ ELISpot kit (Diaclone, USA) was used. Microtiter plates (96-well, Diaclone, USA) were coated overnight with anti-IFN-γ capture antibody according to the manufacturer’s instructions and aspecific binding was blocked with use of TC-1 cell line culture medium for 2 h at 37 °C. When working with splenocytes, the CD8⁺ population was firstly enriched using MACS separation kit Immune cells derived from tumours were seeded into microtiter plates without enrichment for the CD8⁺ population. 2*10⁵ cells per well were incubated with or without E7 peptides for 36 h. The spots were developed according to the manufacturer’s instructions. As a positive control, T cells were stimulated with anti-CD3/anti-CD28 beads. Spots were counted using an AID ELISpot reader (Autoimmun Diagnostika GmbH, Strassberg, Germany). The spots forming unit (SFU) values were normalized, based on the percentage of CD8⁺ T cells detected in the cell suspension by flow cytometry.

Spleens were crushed in RPMI medium with 5% foetal calf serum and 5 × 10⁻⁵ M β-mercaptoethanol, and filtered through a 100 µm cell strainer. After removal of blood cells by ACK Lysing Buffer (Invitrogen), the cells were resuspended and the concentration was adjusted at 2.5 × 10⁶ cells/mL. Then splenocytes were restimulated in different conditions (medium alone, Ova_257-264 peptide (5 µg/mL)) in a volume of 200 µL for one day (ELISPOT) or three days (ELISA). For ELISPOT, plates were revealed with supplied reagents (murine IFNγ ELISPOT kit, Diaclone) and spots were counted with the ImmunoSpot® S6 FluoroSpot Line Plate Reader (C.T.L.). For ELISA, the supernatants were collected and assayed for the presence of IFN-γ using a murine IFN-γ kit (eBioscience). In both ELISPOT and ELISA, restimulation with ionomycine (1 µM) and PMA (0.1 µM) was used to control the viability of lymphocytes.