Troponin-I (High sensitivity mouse cardiac Troponin-I ELISA kit, Life Diagnostics, CTNI-1-HSP) and IFN-γ (Mouse IFN-γ DuoSet ELISA, R&D Systems, DY485-05) levels were determined by ELISA according to manufacturer’s recommendations.
Mouse ifn γ duoset elisa
Manufactured by R&D Systems
The Mouse IFN-γ DuoSet ELISA is a quantitative sandwich enzyme-linked immunosorbent assay kit used for the measurement of mouse interferon-gamma (IFN-γ) levels in cell culture supernatants, serum, and plasma samples.
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3 protocols using mouse ifn γ duoset elisa
1
Cardiac Troponin-I and IFN-γ ELISA Assay
2
Generation of BW5147 Cells Expressing H2Kb and OVA-H2Kb
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BW5147 cells expressing H2Kb and OVA-H2Kb were generated using the retroviral vector system, as previously described [30 (link), 31 (link)]. In brief, the full segment of H2Kb and OVA were amplified using PCR, and cloned into pMX retroviral vector at the BamHI and SalI restrictions sites. Retroviral supernatants were generated by transfecting the pCL-Eco retrovirus packaging vector and each pMX vector containing the gene of interest into the 293T cell line. After transduction with 8 μg/mL polybrene, the populations were cloned by limiting dilution or suspended in DMEM containing 1.5% methylcellulose. The highest expression of a single H2Kb-BW5147 cell was selected with the help of an FITC anti-mouse H2Kb antibody (BioLegend). OVA-H2Kb-BW5147 clones were co-cultured with splenocytes from an OVA-immunized mouse. The highest IFN-γ producing clone was selected after screening with a mouse IFN-γ enzyme-linked immunosorbent assay (mouse IFN-γ DuoSet® ELISA, R&D Systems).
3
In Vitro PMEL T-cell Activation
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Peptide-pulsed DC vaccines (2.0 × 104 cells) that were stimulated with Lipofectamine (1 µl/ml) or B16F10 DNA (1 µg/ml) were cocultured with 1.0 × 105 CFSE-labeled PMELs at a 1:5 ratio in RPMI-1640 supplemented with 10% FBS (200 µl/well) in 96-well plates for 72 h. The amount of IFN-γ in the supernatant was measured by the Mouse IFN-γ Duoset ELISA (R&D), and the proliferation index of PMELs was measured by flow cytometry.
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