Mouse ifn gamma duoset elisa kit

Manufactured by R&D Systems

Sourced in United States

The Mouse IFN-gamma DuoSet ELISA kit is a reagent set for the quantitative determination of mouse interferon-gamma levels in cell culture supernatants, serum, and plasma samples. The kit includes the necessary components to perform a sandwich ELISA assay.

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5 protocols using mouse ifn gamma duoset elisa kit

Evaluating T-Cell Activation Against Oncolytic Virus Infection

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CT-2A cells were infected with Delta-24-ACT at an MOI of 100, and 4 hours later, the cells were incubated with recombinant murine IFN-γ (100 IU/mL) for 48 hours. Splenocytes were isolated and cocultured with mock-infected or infected CT-2A cells in a 96-well flat-bottomed plate for 72 hours (ratio 40:1). The supernatant of the wells was collected and analyzed using the mouse IFN-gamma DuoSet ELISA kit (DY485, R&D Systems) following the manufacturer’s instructions. To assess the activation of T cells after viral infection, splenocytes from B6.CgThy1a-Tg(TcraTcrb)8Rest/J (PMEL) mice were isolated and seeded (4×10⁵) in a 96-well flat-bottomed plate coated with an anti-CD3 antibody (clone 145-2 C11, 100314 BioLegend) for 24 hours. CT-2A cells (1×10⁴) were mock infected (control) or infected with either Delta-24-RGD or Delta-24-ACT (MOI of 100). Forty-eight hours later, the cells were incubated with hgp100 (25-33) (1 ng/mL) (RP20344 GenScript) for 2 hours. Splenocytes and cells were cocultured for 3 days. The supernatant was collected after 24 and 48 hours of coculture and analyzed using the mouse IFN-gamma DuoSet ELISA kit (DY485 R&D Systems) following the manufacturer’s instructions.

Puigdelloses M., Garcia-Moure M., Labiano S., Laspidea V., Gonzalez-Huarriz M., Zalacain M., Marrodan L., Martinez-Velez N., De la Nava D., Ausejo I., Hervás-Stubbs S., Herrador G., Chen Z., Hambardzumyan D., Patino Garcia A., Jiang H., Gomez-Manzano C., Fueyo J., Gállego Pérez-Larraya J, & Alonso M. (2021). CD137 and PD-L1 targeting with immunovirotherapy induces a potent and durable antitumor immune response in glioblastoma models. Journal for Immunotherapy of Cancer, 9(7), e002644.

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Quantifying Interferon-gamma in Murine Brain Tumor

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Supernatants from homogenized whole brains of naïve mice, untreated or treated GL261-luc2 tumor-bearing mice were assayed for immunocytokine representation using the Mouse IFN-gamma DuoSet ELISA kit (R&D Systems #DY485–05, #DY008). This was conducted in accordance with manufacturer’s instructions.

Sheybani N.D., Witter A.R., Garrison W.J., Miller G.W., Price R.J, & Bullock T.N. (2021). Profiling of the Immune Landscape in Murine Glioblastoma following Blood Brain/Tumor Barrier Disruption with MR Image-guided Focused Ultrasound. Journal of neuro-oncology, 156(1), 109-122.

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ILC2-Mediated CD8+ T Cell Proliferation

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For the proliferation assay, OT-I TCR-transgenic CD8⁺ T cells were isolated and purified as described above, and then labeled with CellTrace dye. CD8⁺ T cells were washed with PBS and stained with 1 µM CTV dye (Invitrogen) in PBS for 20 min at 37°C. The dye was removed by washing five times with culture medium containing 2% FBS. A fixed number of CTV-labeled OT-I CD8⁺ T cells (5 × 10⁴) were mixed with IL-33 activated ILC2s (5 × 10^{3 (link)} at an ILC2/T-cell ratio of 1:10 in 96-well plates in complete RPMI-1640 medium containing 10% FBS, 2 mM L-glutamine, 1% penicillin/streptomycin (Gibco), 100 U/ml IL-2, 20 ng/ml IL-7, and 10 ng/ml IL-15 (PeproTech). In some experiments, OVA protein (1 mg/ml) or OVA peptide 257–264 (10 µg/ml) was added to the coculture system. For cytokine detection, supernatants from co-cultures of antigen-stimulated ILC2-CD8⁺ T cells were harvested after 72 h of culture. The levels of cytokines in the culture supernatants were measured by ELISA (mouse IFN-gamma DuoSet ELISA kit, R&D system, DY485, mouse TNF-alpha DuoSet ELISA kit, R&D system, DY410). And the GZMB production was detected by flow cytometry after 72 h of culture (clone NGZB, Invitrogen).

Wen J., Cheng S., Wang R., Huang Y., Xu L., Ma L., Ling Z., Xu J., Zhao D., Zhang Y, & Sun B. (2023). Group 2 innate lymphoid cells boost CD8+ T-cell activation in anti-tumor immune responses. Oncoimmunology, 12(1), 2243112.

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Cytokine Detection in Cell Supernatants

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The cytokines in the culture supernatants were detected with Mouse IL-17 DuoSet ELISA Kit (R&D Systems, Minneapolis, MN, USA) and Mouse IFN-gamma DuoSet ELISA Kit (R&D Systems) according to the manufacturer’s instructions. Briefly, 96-well plates were coated with the capture antibodies overnight at room temperature. After washing and blocking, the diluted supernatants and recombinant cytokine standards were added to the plates and incubated for 2 h at room temperature. Then, the plates were incubated sequentially with the detection antibodies and streptavidin-HRP, as well as Substrate Reagent (R&D Systems) and Stop Solution. The absorbance was measured at 450 nm with wavelength correction set to 540 nm.

Chen S., Ma B., Li X., Zhang K., Wei Y., Du B., Liu X., Wei R., Li X, & Nian H. (2022). MYC-mediated silencing of miR-181a-5p promotes pathogenic Th17 responses by modulating AKT3-FOXO3 signaling. iScience, 25(10), 105176.

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Colon Protein Extraction and Cytokine Profiling

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The colon from mice was collected, carefully washed with saline, and protein extracted using Lysis Buffer (Tris–HCl 50 mM, NaCl 150 mM, EDTA 5 mM, and Triton-X100 1%) and Cocktail Protease Inhibitor (04,693,159,001, Roche). The expression of IL-12, TNF-α, IFN-γ, IL-1β, IL-10, and IL-33 in the colon were assayed by ELISA according to the manufacturer’s instructions (Mouse TNF-α ELISA Ready-Set-Go, eBioscience, 88–7324–88; Mouse IL-1β ELISA Ready-SET-Go, eBioscience, 88–7013–88; Mouse IL-12/IL-23 total p40 ELISA Ready-SET-Go, eBioscience, 88–7120–88; Mouse IL-10 ELISA Ready-SET-Go 2° Generation, eBioscience, 88–7105–88; Mouse IL-33 DuoSet ELISA kit, R&D Systems, DY3626; Mouse IFN-gamma DuoSet ELISA kit, R&D Systems, DY485).

Corrêa R., de Oliveira Santos I., Braz-de-Melo H.A., de Sant’Ana L.P., das Neves Almeida R., Pasquarelli-do-Nascimento G., Prado P.S., Kobinger G.P., Maurice C.F, & Magalhães K.G. (2021). Gut microbiota modulation induced by Zika virus infection in immunocompetent mice. Scientific Reports, 11, 1421.

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