Elisa max deluxe set mouse ifn γ

These ELISAs were performed in similar fashion as described above according to the manufacturer's protocol (MPO: MPO, Mouse, ELISA kit, Hycult Biotech, HK210-02; IFN-γ: ELISA MAX™ Deluxe Set Mouse IFN-γ, BioLegend, 430804; TFN-α: ELISA MAX™ Deluxe Set Mouse TFN-α, BioLegend, 430904).

Splenic CD4+ T cells from 4.1-NOD.Rag2^–/– or NOD mice were purified with CD4 (LT34) micro-beads (Miltenyi) and activated for 48 h with Dynabeads™ Mouse T-Activator CD3/CD28 (Gibco) in complete T-cell medium (RPMI-1640) (Hyclone) supplemented with 50 µM 2-mercaptoethanol (Sigma), 10% FBS, 2 mM L-glutamine, 1 mM sodium pyruvate, 1% penicillin/streptomycin, and 30 IU/ml hIL-2 (R&D). After stimulation, dynabeads were removed and expansion was maintained for 6–7 days with 100 IU/ml hIL-2 in complete T-cell medium.
Expanded cells were harvested and cocultured, in a U-bottom 96-well plate, with freshly isolated and red blood cell-lysed 10⁵ NOD splenocytes and 0.0001–10 µg/ml peptide in complete T-cell medium supplemented with 30 IU/ml of hIL-2. Cell culture supernatants were collected after 48 h and IFNγ concentrations were determined by ELISA using ELISA MAX™ Deluxe Set Mouse IFN-γ (BioLegend) according to the manufacturer’s instructions. Synthetic soluble peptides (purity >80%) were obtained from GenScript^®.

CD8⁺ T cells were isolated from OT-I spleens and LN using mouse CD8a (Ly-2) MicroBeads (#130-117-044, Miltenyi Biotec), and then labeled with 1 μM of the proliferation and cell tracking dye CFSE (#423801, BioLegend). 1 × 10⁵ labeled CD8⁺ cells were co-cultured with an equal number of PLGA particle loaded DCs or splenocytes of PLGA-MP immunized mice in complete medium. After 72 h, cells were recovered, and OT-I proliferation was measured by CFSE dye dilution of CD8⁺ T cells using flow cytometry. Additionally, IFNγ secretion of culture supernatants was measured by mouse IFNγ ELISA kit (ELISA MAX™ Deluxe Set Mouse IFN-γ, #430804, BioLegend) according to the manufacturer’s protocol.

The conditioned medium was collected from MyxV-infected murine cancer cells and stored at −80 °C until use. Mouse IFN-γ in the conditioned media was quantified using the ELISA MAX™ Deluxe Set Mouse IFN-γ (Biolegend, San Diego, CA, USA) according to the manufacturer’s protocol.

Spleens were collected from infected mice and processed as described above to obtain a single cell suspension. Red blood cells were lysed using commercial buffer (BioLegend) per the manufacturer’s instructions. Cells were resuspended in complete Gibco Dulbecco’s Modified Eagle Medium (DMEM, Gibco, Invitrogen) supplemented with 10% FBS, 10 mM HEPES and 50 μM β-mercaptoethanol, then plated at 2 × 10⁵ cells/well and incubated with or without 3 μg of LdAg. Concanavalin A (Con A, ThermoFisher) was used at 2.5 μg/ml to make positive controls. Supernatants were collected after 72 hours and analyzed using the ELISA Max Deluxe Set Mouse IFN-γ from BioLegend.

3 days after different treatments, the blood samples were collected and coagulated at room temperature for 30 min. Serum was then collected after centrifugation at 15,000 g for 15 min. IFNγ and TNFα were detected using ELISA MAX Deluxe Set Mouse IFNγ and TNFα (BioLegend), respectively.