Mouse ifn gamma quantikine elisa kit

Manufactured by R&D Systems

Sourced in United States

The Mouse IFN-gamma Quantikine ELISA Kit is a quantitative sandwich enzyme immunoassay designed to measure mouse interferon-gamma (IFN-gamma) levels in cell culture supernates, serum, and plasma.

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21 protocols using mouse ifn gamma quantikine elisa kit

Cytokine Profiling in Murine Samples

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BAL samples were obtained as described above. Blood samples were obtained via cardiac puncture and collected in heparinized tubes [14 (link),15 (link)]. Cytokines determinations were performed in serum and BAL samples using commercial ELISA kits: IFN-γ (Mouse IFN-gamma Quantikine ELISA Kit, sensitivity: 2 pg/mL), and IL-10 (Mouse IL-10 Quantikine ELISA Kit, sensitivity: 5.2 pg/mL) from R&D Systems (Minneapolis, MN, USA), TNF-α (Mouse TNF alpha ELISA Kit, sensitivity: 9.1 pg/mL), IL-27 (Mouse IL-27 p28/IL30 Quantikine ELISA Kit, sensitivity: 4.7 pg/mL), IL-17 (Mouse IL-17 Quantikine ELISA Kit, sensitivity 5 pg/mL), IL-6 (Mouse IL-6 Quantikine ELISA Kit, sensitivity: 1.8 pg/mL), IL-8 mouse homolog chemokine KC (Mouse CXCL1/KC DuoSet ELISA, sensitivity 2.3 pg/mL), IL-1β (Mouse IL-1 beta ELISA Kit (ab197742), sensitivity: 1 pg/mL) and MCP-1 (Mouse MCP1 ELISA Kit (ab208979), sensitivity: 0.487 pg/mL) from Abcam (Cambridge, UK).

Dentice Maidana S., Imamura Y., Elean M., Albarracín L., Nishiyama K., Suda Y., Kurata S., Jure M.Á., Kitazawa H, & Villena J. (2023). Oral Administration of Lacticaseibacillus rhamnosus CRL1505 Modulates Lung Innate Immune Response against Klebsiella pneumoniae ST25. Microorganisms, 11(5), 1148.

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Stimulation of Murine Splenocytes

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Splenocytes from the C57BL/6 mice were resuspended in RPMI1640 (2% FBS) with PHA (Ebioscience 00497703) plus indicated cytokines. The IL12, IL2, and GMCSF were purchased from Peprotech. Cells were plated onto 96 well plates (6 × 10⁵/well for experiments in Figure 4 and 2 × 10⁵/well for experiments in Figure 6) and cultured for 24 h. The supernatants were collected, and their levels of IFNγ were measured using the Mouse IFN‐gamma Quantikine ELISA Kit (R&D systems).

Zhang J, & Zhao X. (2021). Administration of fusion cytokines induces tumor regression and systemic antitumor immunity. MedComm, 2(2), 256-268.

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IFN-γ ELISA of Antigen-Specific T Cells

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0.5x 10⁶ cells from tumor-draining lymph nodes were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, L-glutamine (2 mmol/L), penicillin (100 U/mL), streptomycin (100 μg/mL), and 2-mercapthoethanol (2.5x10⁻⁵ mol/L) in a 48-well plate. Naïve BALB/c mice provided the source for 3x10⁶ irradiated (12 Gy) feeder splenocytes pre-loaded with tumor-associated immunodominant antigen AH-1-A5 (SPSYAYHQF)(22 (link)) or irrelevant peptide pMCMV (YPHFMPTNL) used at a final concentration of 1μg/mL (Genscript, Piscataway, NJ). Supernatants were collected after 48 hours, and secreted IFNγ was measured using Mouse IFN-gamma Quantikine ELISA Kit (R&D Systems, Minneapolis, MN). As per manufacturer’s protocol, 50 μL of undiluted supernatant, standards, and recombinant mouse IFN-γ (positive control) were assayed in triplicate wells. Optical densities were determined on a FlexStation 3 plate reader (Molecular Devices, San Jose, CA) set at 450 nm with 540 nm subtraction and standard curve generated using a log/log curve-fit.

Pilones K.A., Charpentier M., Garcia-Martinez E., Daviaud C., Kraynak J., Aryankalayil J., Formenti S.C, & Demaria S. (2020). Radiotherapy cooperates with IL15 to induce antitumor immune responses. Cancer immunology research, 8(8), 1054-1063.

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Myeloid Cell-Mediated CD8+ T Cell Activation

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Myeloid cells were isolated from MC38 tumors after treatment with isotype or α41BB antibodies using EasySep Mouse CD11b Positive Selection Kit II (STEMCELL Technologies). CD8 T cells were isolated from the spleens of OT-I mice using EasySep Mouse CD8 T cell Isolation Kit (STEMCELL Technologies). OT-I T cells were labeled with CellTrace Violet (Invitrogen) prior to co-culture. OT-I T cells were co-cultured with myeloid cells in media containing 1μg/mL OVA_(257–264) peptide with or without neutralizing antibodies for IL-10 (JES5–2A5) or IL-12-p75 (R2–9A5) (both from BioXCell) at a concentration of 10μg/mL each. Cells and supernatants were harvested after 72hrs incubation. IFN-gamma in supernatants were measured by (Mouse IFN-gamma Quantikine ELISA Kit, R&D Systems).

Innamarato P., Asby S., Morse J., Mackay A., Hall M., Kidd S., Nagle L., Sarnaik A.A, & Pilon-Thomas S. (2020). Intratumoral activation of 41BB co-stimulatory signals enhances CD8 T cell expansion and modulates tumor-infiltrating myeloid cells. Journal of immunology (Baltimore, Md. : 1950), 205(10), 2893-2904.

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Isolated Tumor-Infiltrating Lymphocyte Activation

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TILs from mice with MC38 tumors were isolated after treatment with isotype or α41BB antibodies using EasySep Mouse CD90.2 Positive Selection Kit II (STEMCELL Technologies). TILs were cultured in round-bottom 96 well plates with immobilized anti-CD3 antibodies (145–2C11 BD Biosciences) at a concentration of 5μg/mL or with irradiated tumor cell lines. MC38 or irrelevant target B16 tumor cells were exposed to X-ray irradiation at a dose of 2×10⁴ rad and cultured with CD90.2⁺ TILs at a 1:10 (target:TIL) ratio for 48hrs. Supernatants were collected and IFN-gamma was measured by (Mouse IFN-gamma Quantikine ELISA Kit, R&D Systems).

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Cytokine Profiling in Murine Lung and Serum

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Blood samples were obtained by cardiac puncture and collected in heparinized tubes [36 (link),60 (link)]. BAL samples were obtained according to the technique developed in the Laboratory Immunobiotechnology of CERELA-CONICET (San Miguel de Tucuman, Argentina) [38 (link),61 (link)]. The trachea was exposed and intubated with a catheter, and then two sequential bronchoalveolar lavages were performed on each mouse by injecting sterile PBS. The recovered fluid was centrifuged for 10 min at 900× g and the fluid frozen at −70 °C for subsequent cytokine determinations. Tumor necrosis factor alpha (TNF-α), interferon gamma (IFN-γ), interleukin 10 (IL-10), IL-1β, IL-6, IL-17, KC, and monocyte chemoattractant protein 1 (MCP-1) concentrations were determined in serum and BAL samples using commercial ELISA kits. IFN-γ (Mouse IFN-gamma Quantikine ELISA Kit, sensitivity: 2 pg/mL), IL-10 (Mouse IL-10 Quantikine ELISA Kit, sensitivity: 5.2 pg/mL), IL-1β (Mouse IL-1β DuoSet ELISA, sensitivity: 1.5 pg/mL), IL-6 (Mouse IL-6 Quantikine ELISA Kit, sensitivity: 1.8 pg/mL), and IL-17 (Mouse IL-17 Quantikine ELISA Kit, sensitivity: 4.7 pg/mL) from R&D Systems (USA). TNF-α (Mouse TNF alpha ELISA Kit, sensitivity: 9.1 pg/mL), MCP-1 (Mouse MCP1 ELISA Kit, sensitivity: 0.487 pg/mL), and KC (Mouse KC ELISA Kit, sensitivity: 1.95 pg/mL) kits were obtained from Abcam (Cambridge, UK).

Albarracin L., Ortiz Moyano R., Vargas J.M., Andrade B.G., Cortez Zamar J., Dentice Maidana S., Fukuyama K., Kurata S., Jure M.Á., Kitazawa H, & Villena J. (2022). Genomic and Immunological Characterization of Hypermucoviscous Carbapenem-Resistant Klebsiella pneumoniae ST25 Isolates from Northwest Argentina. International Journal of Molecular Sciences, 23(13), 7361.

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IFN-γ ELISPOT Assay for Splenocyte Responses

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IFN-γ ELISPOT assay was performed by culturing 2 × 10⁵ splenocytes per well in IFN-γ ELISPOT plates (MAHAS45; Millipore, Molsheim, France). Five peptide epitopes of hSGCA with the highest probability of presentation by H-2Kb MHC class I molecules were identified by the Immune Epitope Database (http://www.iedb.org) and synthesized by GeneCust. Cells were stimulated either with one of these peptides or with 1 μM hSGCA-GST recombinant protein or with SIINFEKL peptide. As a positive control, cells were stimulated with 5 μg/mL Concanavalin A (Sigma Aldrich, St. Louis, MO, USA). After 24 h of culture at 37°C, plates were washed and the secretion of IFN-γ was revealed with a biotinylated anti-IFN-γ antibody (eBioscience, Thermo Fisher Scientific, Waltham, MA, USA), Streptavidin-Alkaline Phosphatase (Roche Diagnostics, Mannheim, Germany), and BCIP/NBT substrate (Mabtech, Les Ulis, France). Spots were counted using an AID reader (Cepheid Benelux, Leuven, Belgium) and the AID ELISpot Reader v6.0 software. Spot-forming units (SFUs) per million cells were represented after subtraction of background values obtained with unstimulated splenocytes.
IFN-γ secreted by splenocytes stimulated with the recombinant protein hSGCA-GST was measured with the Mouse IFN-gamma Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA).

Poupiot J., Costa Verdera H., Hardet R., Colella P., Collaud F., Bartolo L., Davoust J., Sanatine P., Mingozzi F., Richard I, & Ronzitti G. (2019). Role of Regulatory T Cell and Effector T Cell Exhaustion in Liver-Mediated Transgene Tolerance in Muscle. Molecular Therapy. Methods & Clinical Development, 15, 83-100.

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Measuring IFN-γ Secretion from Stimulated Splenocytes

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Splenocytes were cultivated and stimulated in vitro, and the concentrations of IFN-γ in the culture media were measured by a previously described method.¹⁹ (link) Briefly, 4 × 10⁵ splenocyte cells were plated and stimulated with 4 × 10⁴ mitomycin C-treated MBT-2 cells for 3 days. Then the supernatant was collected and frozen at −80°C for the ELISA assay. The secretion of IFN-γ in the supernatant was measured by the Mouse IFN-gamma Quantikine ELISA Kit (R&D Systems, Minneapolis, MN, USA). The procedures were carried out according to the manufacturer’s protocol.

Kitagawa K., Tatsumi M., Kato M., Komai S., Doi H., Hashii Y., Katayama T., Fujisawa M, & Shirakawa T. (2021). An oral cancer vaccine using a Bifidobacterium vector suppresses tumor growth in a syngeneic mouse bladder cancer model. Molecular Therapy Oncolytics, 22, 592-603.

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Bronchoalveolar Lavage Fluid Analysis

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Bronchoalveolar lavage (BAL) samples were obtained as described previously [10 (link)]. Briefly, the trachea was exposed and intubated with a catheter, and 2 sequential lavages were performed in each mouse by injecting sterile PBS. The recovered fluid was centrifuged and frozen at −70 °C for subsequent analyses. Albumin content, a measure to quantitate increased permeability of the bronchoalveolar–capillary barrier, and lactate dehydrogenase (LDH) activity, an indicator of general cytotoxicity, were determined in the acellular BAL fluid as described previously [10 (link),18 (link)].
Interferon (IFN)-β (Mouse IFN-beta ELISA Kit, sensitivity: 15.5 pg/mL), IFN-γ (Mouse IFN-gamma Quantikine ELISA Kit, sensitivity: 2 pg/mL) and IL-10 (Mouse IL-10 Quantikine ELISA Kit, sensitivity: 5.2 pg/mL) concentrations in BAL samples were measured with commercially available enzyme-linked immunosorbent assay (elisa) technique kits following the manufacturer’s recommendations (R&D Systems, Minneapolis, MN, USA).

Raya Tonetti F., Clua P., Fukuyama K., Marcial G., Sacur J., Marranzino G., Tomokiyo M., Vizoso-Pinto G., Garcia-Cancino A., Kurata S., Kitazawa H, & Villena J. (2022). The Ability of Postimmunobiotics from L. rhamnosus CRL1505 to Protect against Respiratory Syncytial Virus and Pneumococcal Super-Infection Is a Strain-Dependent Characteristic. Microorganisms, 10(11), 2185.

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Profiling Tumor-Infiltrating CD8+ T Cells

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For detection of granzyme B expression of infiltrated CD8⁺ T cells, tumour tissues were extracted three days after the last injection and dissociated to single cells using a gentleMACSTM Octo Dissociator (Miltenyi Biotec). CD8⁺ T cells were selected using CD8a T cell isolation kit (Miltenyi Biotec). The isolated CD8⁺ T cells were pre-incubated with 2 μg anti-mouse CD16/CD32 (BD Biosciences) for 5 min at 4°C and then dyed with FITC-anti-CD8α (clone 53–6.7, 100705) and PerCP/Cy5.5-anti-Granzyme B (clone QA16A02, 372211) antibodies. Data were obtained through an Accuri^TM C6 Flow Cytometer and analysed with the FlowJo-V10 software (BD Biosciences).
For detection of tumour-specific T cell activity, spleens were harvested three days after the last injection and dissociated to single cells using a gentleMACS^TM Octo Dissociator (Miltenyi Biotec). The single cell suspensions were incubated with 10 μg/ml Ova peptide (SIINFEKL) for 48 h and the quantity of IFN-γ in each supernatant was analysed through a mouse IFN-gamma Quantikine ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Hong Y., Kim Y.K., Kim G.B., Nam G.H., Kim S.A., Park Y., Yang Y, & Kim I.S. (2019). Degradation of tumour stromal hyaluronan by small extracellular vesicle-PH20 stimulates CD103+ dendritic cells and in combination with PD-L1 blockade boosts anti-tumour immunity. Journal of Extracellular Vesicles, 8(1), 1670893.

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