Mouse ifn γ elispot kit

CD8 T cells were isolated from a single-cell suspension of splenocytes prepared from KPC-N tumor-bearing mice, using CD8 + T cell Isolation Kit (Miltenyi Biotec). For in vitro stimulation, 1 × 10⁵ CD8 T cells were cocultured with 1 × 10⁴ KPC-N target cells in 96 wells plate for 24 h at 37 °C. 1 × 10⁴ MC38 cells were used as negative control to assess the target specificity. IFN-γ-producing cells were quantified using Mouse IFN-γ ELISPOT kit (R&D Systems) according to the manufacturer’s instructions. IFN-γ spots were counted on ImmunoSpot S6 Analyzer (Cellular Technology Limited).

The assays were performed using the mouse IFN-γ ELISpot kit (R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer’s instructions. Splenocytes were plated (100 µL/well) in duplicate 5 × 10⁶ cells/mL and stimulated by a mixture of peptides (corresponding to VACV-specific BALB/c mice H2-d restricted epitopes): SPYAAGYDL, SPGAAGYDL, VGPSNSPTF, KYGRLFNEI, GFIRSLQTI, and KYMWCYSQV [16 (link),23 (link)]. Pooled peptides (100 µL/well) were added at a concentration of 20 µg/mL for each peptide. Non-stimulated and concanavalin A (Con A, 5 μg/mL) stimulated splenocytes were used as negative and non-specific positive controls, respectively. After an 18-h stimulation period at 37 °C in 5% CO₂, cells were discarded, and plates were incubated for 2 h at 37 °C with anti-IFN-γ detection antibodies.
Plates were washed and the spots were revealed by adding the streptavidin-conjugated alkaline phosphatase and BCIP/NBT (5-bromo-4-chloro-3′-indolylphosphate/nitro-blue tetrazolium) substrate. The reaction was stopped by washing plates with distilled water. The number of IFN-γ-producing cells was counted using an ELISpot reader (Carl Zeiss, Jena, Germany).

Eight or nine days after the second booster, mice (3–4 mice/group) were euthanized with Pentobarbitone sodium (100–150 mg/kg, i.p.) and spleens were harvested, homogenised and forced through 70 μm cell strainer (BD) with the ends of sterile syringe plungers [45 (link)]. The splenocytes were obtained by centrifugation at 250 g. Erythrocytes were lysed with ACK buffer for 5 min at room temperature. After washing once with PBS, splenocytes were resuspended in RPMI 1640 medium with 10% FBS, and filtered with 70 μm cell strainer (BD) to get rid of cell aggregates.
Interferon-gamma (IFN-γ) and Interleukine-17A (IL-17A) ELISPOT assays were conducted with Mouse IFN-γ ELISPOT kit (R&D Systems) and Mouse IL-17 ELISpot Kit (R&D Systems) respectively, according to the manufacturer’s instructions. Briefly, splenocytes, stimulated by antigen rSaEsxA or rSaEsxB at the concentration of 2 μg/mL, were plated at the concentration of 5E + 05 cells/well in duplicate for 20 h at 37 °C. Then plates were washed and incubated with biotinylated anti-IFN-γ or anti-IL-17A antibody overnight at 4 °C. After washing the plates, streptavidin-Alkaline phosphatase was added and incubated for 2 h at room temperature. At last, the plates were incubated with substrate BCIP/NBT Chromogen for 0.5-1 h at room temperature for color development. The spots were counted using an immunospot reader system.

Splenocytes were pooled for each treatment group, and single-cell suspensions were prepared. CD8a⁺ T cells were isolated by negative selection using magnetic beads (Miltenyi Biotec), then stimulated in vitro with irradiated naïve splenocytes and antigen. Specifically, 125,000 isolated T cells were co-incubated with irradiated splenocytes (500,000/well) pulsed with 10μg/ml p15E peptide (KSPWFTTL, H-2K^b, ref. [21 (link)]), 10μg/ml control VSV-NP peptide (RGYVYQGL, H-2K^b), irradiated MC38 cells (25,000/well), or irradiated control EL-4 cells (25,000/well). In vitro stimulation was carried out in 96 well-plates that had been coated with anti-IFN-γ antibody (Mouse IFN-γ ELISPOT kit, R&D Systems, Minneapolis, MN). After 36 hours, the plate was developed according to the kit manufacturer's instructions. Co-cultures were carried out in quadruplicate. Spot counts were analyzed using an ELISpot plate reader (CTL ImmunoSpot, Shaker Heights, OH) and reported as mean ± SE for each treatment group.

C57BL/6 mice (9 weeks old, female) were intramuscularly administered 1 µg OVA mRNA-loaded LNPs into the left thigh muscles twice at 3-week intervals. At 1-week post-boost, the animals were euthanized by cervical dislocation, and the spleens were collected and placed in RPMI 1640 medium (R5886, Sigma-Aldrich, St Louis, MO, USA). The spleen of each animal was dissociated using a gentleMACS Octo Dissociator with Heaters (Miltenyi Biotec, Bergisch Gladbach, Germany), filtered through a 40 µm cell strainer, and washed. The pellets were treated with ACK Lysing buffer (Thermo Fisher Scientific) for 2 min at 25 °C and washed. The cells were resuspended in RPMI 1640 medium supplemented with 10% FBS (10270-160, Gibco, Grand Island, NY, USA) and 1% L-Glutamine-Penicillin-Streptomycin solution (G1146, Sigma-Aldrich). OVA-specific cellular immune responses were investigated using a Mouse IFN-γ ELISPOT Kit (R&D systems, Minneapolis, MN, USA). Cells (3.75×10⁴) were incubated with 10 μM OVA class I peptide (SIINFEKL, Sigma-Aldrich) at a 5% CO₂ atmosphere and 37 °C for 20 h, and IFN-γ secreting cells were visualized according to the manufacturer’s protocol. The spots were counted using an ImmunoSpot S6 Analyzer (Cellular Technology Limited, Cleveland, OH, USA).

For detection of IFNγ release, T cells isolated from TLSs and MC38 tumors were mixed with irradiated MC38 cells at a ratio of 10:1 or not in 96-well plates. Culture supernatants were collected after 24 and 48 h, and IFNγ production was measured with an IFNγ ELISA kit (BD Bioscience). Isolated T cells were seeded at 1.25e5/well, and the number of IFNγ-producing cells was measured using a mouse IFNγ ELISpot Kit (R&D systems). The number of positive spots was enumerated using an automatic ELISPOT counter (AID).