To prepare the target cells, GL261 or GL261-OVA cells were infected with virus at 100 PFU per cell. Four hours later, 100 units/ml of mouse IFNγ (Prospec Protein Specialists) was added to the culture. Forty-eight hours after viral infection, the cells were fixed with 1% paraformaldehyde. To activate immune cells, pre-fixed target cells (2 × 104/well) were incubated with splenocytes (5 × 105/well) or brain-infiltrating lymphocytes (BILs) (5 × 104/well). To measure the OVA-specific T-cell reaction, CD8+ T-cells (3 × 105/well) were stimulated with pre-fixed mouse dendritic cells (1 × 105/well) primed with the OVA 257-264 peptide (InvivoGen). Forty hours after the co-culture in a round-bottom 96-well plate, the concentration of IFNγ in the supernatant was assessed with an ELISA (mouse IFNγ DuoSet, R&D Systems).
Mouse ifnγ
Manufactured by ProSpec
Mouse IFNγ is a recombinant protein that represents the murine interferon gamma cytokine. Interferon gamma is a key regulator of immune responses and plays a crucial role in the activation of macrophages and other immune cells.
Automatically generated - may contain errors
Lab products found in correlation
Ova257 264 peptide, by InvivoGen (1 mentions)
Mouse ifn γ duoset, by R&D Systems (1 mentions)
Mouse ifn γ duoset elisa kit, by R&D Systems (1 mentions)
Ovalbumin (ova), by Thermo Fisher Scientific (1 mentions)
Trp 2, by AnaSpec (1 mentions)
96 well round bottom dishes, by Avantor (1 mentions)
Ifn gamma duoset elisa, by R&D Systems (1 mentions)
3 protocols using mouse ifnγ
1
Activation of OVA-specific T-cells
2
Evaluating IFNγ-Induced Antigen Presentation
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To prepare the target cells, 50 units/mL mouse IFNγ (Prospec Protein Specialists) was added to the B16-OVA cell culture. Forty-eight hours later, the cells were fixed with 1% paraformaldehyde and washed before being added to the coculture. For immune cell stimulation, splenocytes (5 × 105 cells/well) were incubated with prefixed target cells (B16-OVA, 5 × 104 cells/well) or one of the following peptides at 2 μg/mL: mgp100 (refs. 24–32 (link); AnaSpec), OVA (257–264; Invitrogen), TRP-2 (180–188; AnaSpec), or GFP (118–126; Biomatik). Twenty-four hours after incubation in a round-bottom 96-well plate, the concentration of IFNγ in the medium was assessed with a mouse IFNγ DuoSet ELISA kit (R&D Systems).
3
IFNγ-Mediated Immune Cell Activation
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2 × 104 target GL261-5 cells/well were seeded on 96 well-round bottom dishes (VWR) and infected with virus at 100 ifu per cell. Four hours later, 100 units/mL of mouse IFNγ (ProSpec) were added to the culture. Forty-eight hours after viral infection, the cells were detached with 2 mM EDTA in PBS, fixed with 1% paraformaldehyde, then cleaned with Lysine (0.1 M) wash solution. 2 × 104 fixed cells were seeded to 96 well-round bottom dishes (VWR). To activate immune cells, pre-fixed target cells were incubated with splenocytes (5 × 105/well) for 48 hours. Then, the concentration of IFNγ in the supernatant was assessed with standard ELISA (IFN-gamma DuoSet ELISA, R&D system).
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