Ifn gamma mouse uncoated elisa kit

Manufactured by Thermo Fisher Scientific

Sourced in Austria, United States

The IFN gamma Mouse Uncoated ELISA Kit is a laboratory equipment product designed for the quantitative determination of mouse interferon gamma (IFN-γ) levels in biological samples. It is an enzyme-linked immunosorbent assay (ELISA) kit that provides the necessary components to perform this specific measurement.

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10 protocols using ifn gamma mouse uncoated elisa kit

Inflammatory Cytokine Profiling in Acinetobacter Infection

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Serum and peritoneal lavage fluid (PLF) samples were collected from 10 mice/group infected with A. baumannii and treated with FT, as described previously. The IL-6 assay range was 31.3–2000 pg ml⁻¹ (determined by the lower limit of detection and the highest point of the standard curve). IL-6 levels in serum and PLF samples were analysed by ELISA (enzyme-linked immunosorbent assay) according to the manufacturer’s instructions (Mouse IL-6 ELISA kit, Invitrogen by Thermo Fisher Scientific, Vienna, Austria). IL-6 levels in serum and PLF were determined in mice at 2, 4, and 14 h p.i. The levels of the cytokines IFNγ (IFN gamma Mouse Uncoated ELISA Kit, Invitrogen by Thermo Fisher Scientific, Vienna, Austria) and TNFα (ELISA MAX™ Deluxe Set Mouse TNF-α, BioLegend, San Diego, USA) in serum and PLF were also assessed 2 h p.i. Optical densities were measured using a Multiskan EX plate reader at a wavelength of 450 nm.

Bondareva N.E., Soloveva A.V., Sheremet A.B., Koroleva E.A., Kapotina L.N., Morgunova E.Y., Luyksaar S.I., Zayakin E.S, & Zigangirova N.A. (2022). Preventative treatment with Fluorothiazinon suppressed Acinetobacter baumannii-associated septicemia in mice. The Journal of Antibiotics, 75(3), 155-163.

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Quantifying Cytokine Secretion in Cells

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Cell-free supernatants were assayed for IFNγ protein concentrations via ELISA using the IFN gamma Mouse Uncoated ELISA Kit (Invitrogen) according to the manufacturer’s protocol and measured in a Tecan infinite M200 platereader. For the quantification of murine IL-2 protein concentration in cell-free supernatants, the ELISA MAX™ Standard Set Mouse IL-2 (Biolegend) was used according to the manufacturer’s protocol and measured in a Tecan infinite M200 platereader. It was experimentally verified that this kit was specifically detecting murine IL-2 but not recombinant human IL-2 that was added as a media supplement (cf. 2.5).

Matthe D.M., Dinkel M., Schmid B., Vogler T., Neurath M.F., Poeck H., Neufert C., Büttner-Herold M, & Hildner K. (2023). Novel T cell/organoid culture system allows ex vivo modeling of intestinal graft-versus-host disease. Frontiers in Immunology, 14, 1253514.

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Evaluating T Cell Activation by Antigen-Pulsed DCs

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Bone marrow cells isolated from the hindlimb bones of C57BL/6J mice were cultured in DC medium for 8 days before harvesting the bone marrow-derived DCs (BMDCs). The cells were incubated with 10 µg/mL SIINFEKL (an ovalbumin (OVA)-derived antigen) at 37°C for 2 hours. Then, the SIINFEKL-pulsed DCs were harvested by centrifugation and resuspended at a density of 3×10⁶ cells/mL in DC medium.
CD8⁺ OT-I T cells were isolated from spleens and cervical lymph nodes of Rag1 KO / transgenic OT-I T Cell Receptor mice (B6.129S7-Rag^1tm1MomTg(TcraTcrb)1100Mjb; OT-I 5.2, Taconic) and purified using the CD8a+T Cell Isolation Kit II (Miltenyi Biotech, Bergisch Gladbach, Germany). BMDCs and CD8⁺ T cells were cocultured (1:1) and treated with 3 nM–3 µM BAY 2416964 in the presence of 100 µM KA. After 72 hours, IFN-γ secretion was analyzed using the IFN gamma Mouse Uncoated ELISA Kit (Thermo Fisher Scientific). Coculture with OT-I T cells and SIINFEKL-pulsed DCs with or without KA treatment served as negative and positive controls, respectively.

Kober C., Roewe J., Schmees N., Roese L., Roehn U., Bader B., Stoeckigt D., Prinz F., Gorjánácz M., Roider H.G., Olesch C., Leder G., Irlbacher H., Lesche R., Lefranc J., Oezcan-Wahlbrink M., Batra A.S., Elmadany N., Carretero R., Sahm K., Oezen I., Cichon F., Baumann D., Sadik A., Opitz C.A., Weinmann H., Hartung I.V., Kreft B., Offringa R., Platten M, & Gutcher I. (2023). Targeting the aryl hydrocarbon receptor (AhR) with BAY 2416964: a selective small molecule inhibitor for cancer immunotherapy. Journal for Immunotherapy of Cancer, 11(11), e007495.

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ELISA Quantification of IFN-γ and IL-10

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IFN-γ and IL-10 concentrations in cell culture supernatants were quantified by using the IFN gamma Mouse Uncoated ELISA Kit and the IL-10 Mouse Uncoated ELISA Kit (both Thermo Fisher Scientific), respectively, according to the manufacturer´s protocol. Samples were measured with the SUNRISE Absorbance Reader and analyzed utilizing the Magellan software (both Tecan Group, Männedorf, Switzerland).

Bauer V., Ahmetlić F., Hömberg N., Geishauser A., Röcken M, & Mocikat R. (2021). Immune checkpoint blockade impairs immunosuppressive mechanisms of regulatory T cells in B-cell lymphoma. Translational Oncology, 14(9), 101170.

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Cytokine Expression Quantification

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IL-12, IL-10, TGF-β, and IFN-γ were quantified using an IL-12 p70 Mouse Uncoated ELISA Kit (Thermo Fisher Scientific), IL-10 Mouse Uncoated ELISA Kit (Thermo Fisher Scientific), TGF beta-1 Human/Mouse Uncoated ELISA Kit (Thermo Fisher Scientific), and IFN gamma Mouse Uncoated ELISA Kit (Thermo Fisher Scientific), respectively, according to the manual of each kit.

Kajiyama S., Nagatake T., Ishikawa S., Hosomi K., Shimada Y., Matsui Y, & Kunisawa J. (2023). Lentinula Edodes Mycelia extract regulates the function of antigen-presenting cells to activate immune cells and prevent tumor-induced deterioration of immune function. BMC Complementary Medicine and Therapies, 23, 281.

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Evaluating Combination Immunotherapy in Murine Tumor Model

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LL/2 tumor-bearing C57BL/6 mice (female, n = 5) were randomly assigned and treated with free drugs, NPs, and/or agonists on a Q4D × 4 schedule. Sequential doses were given as follows: 1 mg Podo/kg (i.v. injection on day 1 of each cycle), 3 mg Carb/kg (i.v. injection on day 3 of each cycle), and/or 30 μg αCD40/mouse (intraperitumoral injection on day 3 of each cycle). Then, the mice were euthanized, their tumors were quickly excised, and the proteins from the tumors were extracted with RIPA Lysis and Extraction Buffer, supplemented with Protease Inhibitor Cocktail (Sigma-Aldrich) and phenylmethylsulfonyl fluoride (PMSF, Sigma-Aldrich). VEGF, endostatin, and IFN-γ concentrations in tumor lysates were measured according to the manufacturer protocols (Mouse VEGF Quantikine ELISA Kit, R&D Systems; Mouse Endostatin ELISA Kit, LifeSpan BioSciences; IFN gamma Mouse Uncoated ELISA Kit, Thermo Fisher Scientific) with a microplate reader.

Ling X., Jiang X., Li Y., Han W., Rodriguez M., Xu Z, & Lin W. (2020). Sequential Treatment of Bioresponsive Nanoparticles Elicits Antiangiogenesis and Apoptosis and Synergizes with a CD40 Agonist for Antitumor Immunity. ACS nano, 15(1), 765-780.

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Evaluating Tumor Immune Responses

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CT26 tumor-bearing BALB/c mice (male, n = 5) were randomly assigned and i.v. injected with free drugs or NCP particles at 0.5 mg Dig/kg, 5 mg Carb/kg, and/or 50 nmol siPD-L1/mouse on a Q3D × 5 schedule. Then mice were sacrificed, and their tumors, tumor draining inguinal lymph nodes, and spleens were immediately collected. Single-cell suspensions from lymph nodes were seeded in 96-well plates (2.0 × 10⁵ cells per well) and incubated with complete medium with stimulation of antibodies against CD3ɛ (Invitrogen, 16–0031-85; 1:500) plus CD28 (Invitrogen, 16–0281-85; 1:500). After 72 h incubation, IFN-γ concentrations in supernatants were assessed with an IFN gamma Mouse Uncoated ELISA Kit (Thermo Fisher Scientific, USA) using a microplate reader. IFN-γ concentrations in tumor lysates were valued using IFN gamma Mouse Uncoated ELISA Kit. Single-cell suspensions from spleens were seeded in a capture antibody pre-coated MultiScreen-IP Filter Plate (5 × 10⁵ cells per well) and incubated with complete medium with or without stimulation of SPSYVYHQF peptidic epitope (10 μg/mL, PEPTIDE 2.0, USA). After 48 h incubation, IFN-γ concentrations were tested using Mouse IFN gamma ELISpot (Thermo Fisher Scientific, USA) and AEC Substrate Set (BD, USA), then counted with an Immunospot S6 CORE Analyzer (Cellular Technology, USA).

Ling X., Han W., Jiang X., Chen X., Rodriguez M., Zhu P., Wu T, & Lin W. (2021). Point-source burst of coordination polymer nanoparticles for tri-modality cancer therapy. Biomaterials, 270, 120690.

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Quantification of IFN-γ in Th17 Cells

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IFN-γ in the culture supernatant of in vitro-differentiated Th17 cells stimulated under IFN-γ-inducing conditions was quantified using the IFN gamma Mouse Uncoated ELISA Kit (Thermo Fisher Scientific) as described in the manufacturer’s instructions.

Choi G., Park Y.J., Cho M., Moon H., Kim D., Kang C.Y., Chung Y, & Kim B.S. (2021). A critical role for Th17 cell-derived TGF-β1 in regulating the stability and pathogenicity of autoimmune Th17 cells. Experimental & Molecular Medicine, 53(5), 993-1004.

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NK Cell IFN-γ Production Assay

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1 × 10⁵ NK cells were seeded into a 96-well plate and cultured with or without IL-12 plus IL-18 for 24 h. Supernatants were then harvested to detect IFN-γ production, which was assessed by the Mouse IFN-gamma Uncoated ELISA Kit (Catalog #88-7314-88, Invitrogen)

Wang Y., Dong W., Zhang Y., Caligiuri M.A, & Yu J. (2018). Dependence of innate lymphoid cell 1 development on NKp46. PLoS Biology, 16(4), e2004867.

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In vitro Tumor Cell Labeling with DSPE-PEG-FITC

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For in vitro labelling of tumour cells with amph-FITC, tumour cells were washed with 1× phosphate-buffered saline (PBS) and incubated at a cell density of 10⁶ cells ml⁻¹ for 30 min at 37 °C with DSPE-PEG_x-FITC, where x = 0, 1, 2, 3.4 or 10 kDa (Creative PEGworks) in PBS. For co-cultures, target cells were labelled simultaneously with 100 nM amph-FITC and CellTrace Violet (ThermoFisher); 20,000 target cells were seeded per well in a 96-well flat-bottom plate with CAR T cells at the indicated E:T ratios. Following a 16 h incubation, cells were stained with SYTOX red (ThermoFisher) for flow cytometry, and IFN-γ in the supernatant was quantified using a Mouse IFN gamma Uncoated ELISA Kit (Invitrogen).

Zhang A.Q., Hostetler A., Chen L.E., Mukkamala V., Abraham W., Padilla L.T., Wolff A.N., Maiorino L., Backlund C.M., Aung A., Melo M., Li N., Wu S, & Irvine D.J. (2023). Universal redirection of CAR T cells against solid tumours via membrane-inserted ligands for the CAR. Nature Biomedical Engineering, 7(9), 1113-1128.

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