Mouse ifn γ elisa max

ID8 cells were provided by Gordon Freeman (DFCI). Cells were cultured in DMEM with 10% FBS (Heat inactivated). The rest of the cell lines were received from ATCC and cultured in their recommended media. Splenocytes were cultivated in SILAC RPMI (Thermo # 89984) media supplemented with: 10% Heat Inactivated FBS, Sodium pyruvate 1 mM, Glutamine 1 mM, Penicillin (50 U/ML) Strptomycin (50μg/ml), 2-Mercaptoethanol; 0.00005 M, (Sigma M7522), IL7: 5 ng/ml (PeproTech, #217–17 murine IL7), IL15: 100 ng/ml (PeproTech, #210–15 murine IL15), N-acetyl L Cysteine: 0.5 M (Sigma A9165), Lysine 218 uM and Arginine: 75 uM (or as indicated). Recombinant arginase was purchased from (R&D Biosystems, 5868AR010). Purified anti-mouse CD3 (cat# 100202) and purified anti-mouse CD28 (cat# 102102) antibodies were purchased from BioLegend. IFNg ELISA was performed using Biolegend (Mouse IFN-γ ELISA MAX).

In vitro binding of nanoparticles was assessed after incubation of 250,000 enriched CD8 T cells with fluorescently (DiD, Life Technologies) labeled nanoparticles at different concentrations for 30 min at 37 °C. After the incubation, T cells were washed 3 to 5 times in PBS and directly assessed by flow cytometry for DiD fluorescence. For internalization studies, cells were incubated for 30 min on ice, washed, and either stained immediately with AF488-anti-F(ab’)2 antibody or incubated for the indicated periods of time at 37 °C before secondary staining. AF488-anti-F(ab’)2 secondary antibodies were purchased from Jackson ImmunoResearch Laboratory (product 109-545-097 for anti-human PD-1, 112-545-006 for anti-rat CD8a; both diluted 1:100). T cells isolated from OT-I Rag1^−/− mice were activated by Dynabeads Mouse T-Activator CD3/CD28 (Thermo Scientific) at a ratio of 2:1 T cell to bead or by ova-expressing B16 melanoma cells at a ratio of 10:1 T cell to B16 cell. Carboxyfluorescein succinimidyl ester (CFSE, BioLegend) or Cell Trace Violet (Thermo Scientific) was used to assess T cell proliferation; the labeling was carried out according to the manufacturer’s recommendations. Mouse TGFβ1 was purchased from Cell Signaling Technologies, and T cell supernatants were analyzed by mouse IFNγ ELISA MAX™ (BioLegend).