Elisa max standard set mouse ifn γ

Manufactured by BioLegend

Sourced in United States

The ELISA MAX standard set mouse IFN-γ is a laboratory equipment product used for enzyme-linked immunosorbent assays (ELISA) to detect and quantify mouse interferon-gamma (IFN-γ) in samples. It provides the necessary components, including standards, to perform the ELISA procedure.

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6 protocols using elisa max standard set mouse ifn γ

Cytokine Profiling in Immune Cell Cultures

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Cell culture supernatants were collected from (1) BMDC (100,000 cells/well) cultures 8 hours after stimulation, (2) DC-T cocultures after a 3-day incubation period, or (3) total draining lymph node (dLN) cell cultures (500,000 cells/well) kept in culture for 2 days, and stored frozen at −80°C in single-use aliquots.
For sandwich Enzyme-Linked Immunosorbent Assay (ELISA), cell culture supernatants were diluted using the ELISA assay diluent (Biolegend 4212013) and cytokine concentrations were measured using the ELISA MAX standard set mouse IFN-γ (BioLegend 430801) and IL-17A (BioLegend 432501) kits according to the manufacturer’s instructions.
For flow cytometric, bead-based immunoassays, DC and dLN cell culture supernatants were diluted and processed using the LEGENDplex mouse anti-virus response panel (BioLegend 740622) and the LEGENDplex mouse Th cytokine panel (BioLegend 740740) kits, respectively. Data were acquired on the NovoCyte flow cytometer (Acea Biosciences/Agilent) and analyzed using the LEGENDplex software v8 (BioLegend).

Pandey S., Gruenbaum A., Kanashova T., Mertins P., Cluzel P, & Chevrier N. (2020). Pairwise Stimulations of Pathogen-Sensing Pathways Predict Immune Responses to Multi-Adjuvant Combinations. Cell systems, 11(5), 495-508.e10.

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Detection of Murine Cytokines by ELISA

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After the splenic cells had been stimulated with RBD protein for 42 hours, the culture supernatant was harvested for the detection of mouse cytokines via ELISA (ELISA MAX™ standard Set Mouse IL-2, ELISA MAX™ standard Set Mouse IL-4, ELISA MAX™ standard Set Mouse IL-10, ELISA MAX™ standard Set Mouse IFN-γ, Biolegend). Due to the limited volume, harvested supernatant from one group was pooled, and three parallel wells were set. The following detection limits were obtained from the manufactures: IL-2 (detection limit 2.0 pg/mL), IL-4 (detection limit 2.0 pg/mL), IL-10 (detection limit 31.3 pg/mL), and IFN-γ (detection limit 15.6 pg/mL). Dilution factors of the samples were determined during a preliminary experiment. The experimental ELISA was carried out according to the manufacturer’s instructions.

Chen L., Zhang H., Li M., Wu B., Zhang Z, & Gong R. (2022). An intranasal vaccine targeting the receptor binding domain of SARS-CoV-2 elicits a protective immune response. Frontiers in Immunology, 13, 1005321.

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Quantifying IFN-γ Responses and T-cell Subsets

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The IFN-γ-secreting splenocytes were measured at 7, 17, and 45 dpi using a mouse IFN-γ enzyme-linked immune absorbent spot (ELISpot) assay (BD Life Sciences, USA). Briefly, 5 × 10⁵ splenocytes/well were stimulated with Z_EDIII (1 μg/ml). Spots were counted under a dissecting microscope (Olympus, model no. SZH-ILLB). To measure secreted cytokines, splenocytes (5 × 10⁵/well) were stimulated with Z_EDIII (1 μg/ml) for 48 h and the supernatant of splenocytes was collected. The levels of cytokines were determined using mouse tumor necrosis factor (TNF)-α ELISA MAX™ standard set, mouse IFN-γ ELISA MAX™ standard set, and mouse interleukin (IL)-12 ELISA MAX™ standard set (BioLegend, USA). Each assay was performed in triplicate. To analyze T lymphocyte subtypes, splenocytes were stained with the following antibodies at a dilution of 1:200 with 1% BSA in PBS at 4 °C for 1 h: anti-mouse CD3 (clone 145-2C11, BD Biosciences), and anti-mouse CD4 (clone RM4-5, BD Biosciences) or anti-mouse CD8 (clone 53–6.7, BD Biosciences) as described previously [27 (link)]. Cell phenotype was analyzed using a FACS Calibur flow cytometer (Becton Dickinson).

Shin M., Kim K., Lee H.J., Jung Y.J., Park J, & Hahn T.W. (2022). Vaccination with a Zika virus envelope domain III protein induces neutralizing antibodies and partial protection against Asian genotype in immunocompetent mice. Tropical Medicine and Health, 50, 91.

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Evaluating Cytokine Profiles in Immunized Mice

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The interferon (IFN)-γ secreting splenocytes were measured at 7, 17, and 45 days after the first immunization using mouse IFN-γ enzyme-linked immune absorbent spot (ELISpot) assay (BD Life Sciences, USA). Briefly, 5 × 10⁵ splenocytes/well were stimulated with Env_M (1 μg/ml) or Env_Z (1 μg/ml). Spots were counted under a dissecting microscope (Olympus, model no. SZH-ILLB).
For cytokine analysis, 5 × 10⁵ splenocytes/well were stimulated with Env_M (1 μg/ml) or Env_Z (1 μg/ml). The media from splenocyte cultures were collected after 48 h incubation. The levels of cytokines were determined using mouse tumor necrosis factor (TNF)-α ELISA MAX™ standard set, mouse IFN-γ ELISA MAX™ standard set, and mouse interleukin (IL)-12 ELISA MAX™ standard set (BioLegend, USA). Each assay was performed in triplicate.
To analyze T lymphocyte subtypes, splenocytes were stained with the following antibodies at a dilution of 1:200 with 1% BSA in PBS at 4 °C for 1 h: anti-mouse CD3 (fluorescein isothiocyanate, BD Biosciences), and anti-mouse CD4 (phycoerythrin [PE], BD Biosciences) or anti-mouse CD8 (PE, BD Biosciences). Cell phenotype was analyzed using a FACSCalibur flow cytometer (Becton Dickinson).

Shin M., Kim K., Lee H.J., Lee R., Jung Y.J., Park J, & Hahn T.W. (2022). Zika virus baculovirus-expressed envelope protein elicited humoral and cellular immunity in immunocompetent mice. Scientific Reports, 12, 660.

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Th1 Polarization of Naive CD4+ T-cells

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Naïve CD4⁺ T-cells (1 x 10⁶/ml) from CK2α^fl/fl or CK2α ^fl/fldLck-Cre mice were polarized under Th1 conditions. IFN-γ levels in the supernatant were measured using the MAX™ Standard Set Mouse IFN-γ ELISA (BioLegend, San Diego, CA).

Yang W., Gibson S.A., Yan Z., Wei H., Tao J., Sha B., Qin H, & Benveniste E.N. (2020). Protein Kinase 2 (CK2) Controls CD4+ T-cell Effector Function in the Pathogenesis of Colitis. Mucosal immunology, 13(5), 788-798.

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Th1 Polarization of Naive CD4+ T-cells

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