Anti ifn γ mab

Manufactured by Thermo Fisher Scientific

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The Anti-IFN-γ mAb is a monoclonal antibody specific for interferon-gamma (IFN-γ). It can be used to detect and quantify IFN-γ in various biological samples.

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IFN-γ (1815 citations) Anti-IFN-γ (147 citations) PE-conjugated anti-human IFN-γ mAbs (2 citations)

9 protocols using anti ifn γ mab

Isolation and Differentiation of Murine CD4+ T Cell Subsets

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Splenic naive CD4 T cells (CD44^loCD62L^hi) from C57BL/6, Cxxc1^fl/fl (WT), or Cxxc1^fl/fl ER-Cre⁺ (KO) mice were purified using a magnetic cell sorter (autoMACS; Miltenyi Biotec), yielding a purity of >98%. Purified naive CD4⁺ T cells were stimulated with immobilized anti-TCRβ mAb (H57-597; 10 µg/ml) in the presence of IL-2 (25 U/ml), IL-12 (10 U/ml; BD PharMingen), anti–IL-4 mAb (11B11; 5% vol/vol), and CD28 mAb (37.51; 1 µg/ml) for Th1 cell differentiation; IL-2 (25 U/ml), IL-4 (100 U/ml; PeproTech), anti-IFNγ mAb (R46A2; 5% vol/vol), and CD28 mAb (37.51; 1 µg/ml) for Th2 cell differentiation; TGF-β1 (2.5 ng/ml; PeproTech), IL-6 (25 ng/ml; PeproTech), anti–IL-4 mAb (11B11; 5% vol/vol), anti-IFNγ mAb (R46A2; 5% vol/vol), anti–IL-2 mAb (JES6-1A12; 2.5 µg/ml; BD PharMingen), and CD28 mAb (37.51; 1 µg/ml) for Th17 cell differentiation; and IL-2 (50 U/ml), TGF-β1 (10 ng/ml; PeproTech), anti-IFNγ mAb (R46A2; 5% vol/vol), anti–IL-4 mAb (11B11; 5% vol/vol), and anti-CD28 mAb (37.51; 1 µg/ml) for iT reg cell differentiation. The anti-TCR mAb and anti-CD28 mAb stimulation was stopped at 48 h, after which the cells were cultured with the above-described cytokines. The cells are referred to as Th1 or Th2 cells with prolonged TCR/coreceptor stimulation if the anti-TCR mAb and anti-CD28 mAb stimulation was not stopped at 48 h.

Kiuchi M., Onodera A., Kokubo K., Ichikawa T., Morimoto Y., Kawakami E., Takayama N., Eto K., Koseki H., Hirahara K, & Nakayama T. (2021). The Cxxc1 subunit of the Trithorax complex directs epigenetic licensing of CD4+ T cell differentiation. The Journal of Experimental Medicine, 218(4), e20201690.

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Th1, CTL, and MDSC Identification

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Resuspended PBMCs (1×10⁶) were cultured in 1 ml 10% fetal bovine serum RPMI 1640 medium in the presence of 50 ng/ml PMA (Sigma-Aldrich, California, USA) and 1 μg/ml of ionomycin (Sigma-Aldrich). After 1 hour, 1 μg/ml brefeldin-A (eBioscience, San Diego, USA) was added to the culture for 4 hours at 37℃ and 5% CO2. Stimulated PBMCs were stained with PE-Cy5-conjugated anti-CD3 mAb and FITC-conjugated anti-CD8 mAb (eBioscience), then fixed, permeabilized and stained with anti-IFN-γ mAb (eBioscience) with an Intracellular Staining Kit (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. Th1 were defined as CD3⁺CD8^-IFN-γ⁺, and CTL were defined as CD3⁺ CD8⁺ IFN-γ⁺.
Resuspended PBMCs (1×10⁶) were stained with APC-conjugated anti-HLA-DR mAb (eBioscience), PE-Cy5-conjugated anti-CD11b mAb (Biolegend), FITC-conjugated anti-CD33 mAb (eBioscience), and PE-conjugated anti-ARG-1 mAb (Biolegend); MDSCs were defined as CD33⁺CD11b⁺HLA-DR^-. All data were acquired with a BD FASCalibur flow cytometer and subsequently analyzed with FlowJo software.

Zhou Q., Tang X., Tian X., Tian J., Zhang Y., Ma J., Xu H, & Wang S. (2018). LncRNA MALAT1 negatively regulates MDSCs in patients with lung cancer. Journal of Cancer, 9(14), 2436-2442.

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Investigating HOXA1's Impact on Antitumor Immunity

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To investigate whether HOXA1 could directly impair antitumor immune responses, 0.9 × 10⁶ LLC cells were transplanted into C57BL/6 mice. After a palpable tumor was formed, tumor-bearing mice were treated by intratumoural injections of pcDNA3.1-HOXA1 every 2–3 days for 3 weeks. Tumor volume and weight were measured, and single-cell suspensions from the spleen, draining lymph nodes and tumors of tumor-bearing mice were collected for analysis. Cells were stimulated with PMA (Sigma-Aldrich, St. Louis, MO, USA, 50 ng/mL), ionomycin (eBioscience, San Diego, CA, USA, 1 µg/mL), and monensin (eBioscience, 2 µg/mL) for 5 h. After that, cells were stained with anti-CD3 and anti-CD4/CD8 mAbs (eBioscience), fixed, permeabilized and stained with anti-IFNγ mAb (eBioscience) according to the Intracellular Staining Kit (Invitrogen, Carlsbad, CA, USA) instructions. Cells obtained from tumor tissues were stained with anti-CD11b and anti-Gr1 mAbs (BioLegend). All of the experiments were approved by the Animal Use Committee of Jiangsu University in accordance with the International Guiding Principles for Biomedical Research Involving Animals.

Tian X., Ma J., Wang T., Tian J., Zhang Y., Mao L., Xu H, & Wang S. (2018). Long Non-Coding RNA HOXA Transcript Antisense RNA Myeloid-Specific 1–HOXA1 Axis Downregulates the Immunosuppressive Activity of Myeloid-Derived Suppressor Cells in Lung Cancer. Frontiers in Immunology, 9, 473.

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Phenotyping of Osteogenic Mesenchymal Stem Cells

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To identify the OE-MSCs phenotype, single cell suspensions were stained with relevant fluorochrome-conjugated mouse CD29, CD90, CD44, CD45, CD11b IL-17R mAbs (eBioscience, San Diego, CA). For detection of regulatory T (Treg) cells, anti-CD4, anti-CD25, and anti-Foxp3 mAbs (eBioscience, San Diego, CA) were performed using Foxp3 staining buffer set (eBioscience, San Diego, CA). For intracellular cytokine staining, single cell suspensions were stimulated with phorbol myristateacetate (50ng/ml, SigmaAldrich, St.Louis, MO), ionomycin(1μg/ml, Enzo, Farmingdale, NY) and monensin (2 μg/ml, Enzo, Farmingdale, NY) at 37°C and in a 5% CO₂ atmosphere for 5 hours. Then cells were stained with anti-CD4 mAbs (eBioscience, San Diego, CA), fixed, permeabilized and stained with anti-IFN-γ mAb or anti-IL-17 mAb (eBioscience, San Diego, CA). As controls, the appropriate isotype-matched antibodies were used for each staining. Flow cytometry was performed using a FACS Calibur flow cytometer (eBioscience, San Diego, CA), and the data was analyzed by WinMDI 2.8 software.

Tian J., Rui K., Tang X., Wang W., Ma J., Tian X., Wang Y., Xu H., Lu L, & Wang S. (2016). IL-17 down-regulates the immunosuppressive capacity of olfactory ecto-mesenchymal stem cells in murine collagen-induced arthritis. Oncotarget, 7(28), 42953-42962.

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Analyzing Immune Cell Subsets by Flow Cytometry

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Icariin was purchased from MEYER company (Shanghai, China), whereas the antibodies used for western blotting were purchased from Cell Signaling Technology (Danvers, MA). The antibodies were anti-CD3 mAb (PE), anti-CD4 mAb (APC), anti-CD8a mAb (PE/Cy7), anti-CD25 mAb (APC/Cy7), anti-CD44 mAb (FITC), anti-CD62L mAb (APC/Cy7), anti-IFN-γ mAb (PE), HO-1 mAb (PE/Cy7), anti-CD16/32 mAb and anti-Foxp3 mAb (FITC) (eBioscience, San Jose, CA, USA).

Sai X., Li Z., Deng G., Wang L., Xiaowu W., Nasser M.I., Liu C, & Zhu P. (2022). Immunomodulatory effects of icariin in a myocardial infarction mouse model. Bioengineered, 13(5), 12504-12515.

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Characterization of Activated Cytotoxic T Cells

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CTLs were stained with CD3, CD8 and CD69 anti-human mouse antibodies (mAbs), washed, and analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ). The CD3+ CD8+ cells were gated and evaluated for activation cell subsets (CD69+). Briefly, T cells that were maintained under the same culture conditions but without any peptide stimulation were used as negative controls. CTLs were co-incubated at a 1:1 ratio with stimulator cells in round-bottom 96-well plates. After 1 h of incubation, 10 μg/mL Brefeldin A (eBioscience, San Diego, USA) was added to each well. After 5 h of additional incubation, the cells were centrifuged, washed, and stained with phycoerythrin (PE)-conjugated anti-CD3 (eBioscience) and peridinin-chlorophyll protein (PerCP)-conjugated anti-CD8 anti-human mAbs (Biolegend) for 30 min on ice in the dark. After surface staining, cells were washed, fixed, permeabilized, washed with Perm/Wash solution (BD Biosciences), stained with an anti-IFN-γ mAb (eBioscience), and analyzed by flow cytometry^{38 (link)}.

Xie X., Zhou W., Hu Y., Chen Y., Zhang H, & Li Y. (2018). A dual-function epidermal growth factor receptor pathway substrate 8 (Eps8)-derived peptide exhibits a potent cytotoxic T lymphocyte-activating effect and a specific inhibitory activity. Cell Death & Disease, 9(3), 379.

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T Cell Differentiation Protocols

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Naïve T cells purified from the spleens and lymph nodes of WT or Foxo1 and Foxp1 knockout OT-II mice by FACS (fluorescence-activated cell sorting) sorting or kit (STEMCELL Technologies) were differentiated into T_H1 or T_H9 cells with plate-bound anti-CD3 (2 μg/ml) and soluble anti-CD28 (1 μg/ml) antibodies together with different cytokine combinations: For T_H1 cells, IL-2 (30 ng/ml; R&D Systems), IL-12 (4 ng/ml; R&D Systems), and anti–IL-4 mAb (10 μg/ml; eBioscience) were added; for T_H9 cells, IL-4 (10 ng/ml; R&D Systems), TGF-β (1 ng/ml; R&D Systems), and anti–IFN-γ mAb (10 μg/ml; eBioscience) were added. After 3 days of culture, the differentiated T_H cells were collected, washed, and allowed to continue to differentiate in the presence of the appropriate cytokines for 1 day. For IL-7 treatment, naïve T cells were cultured with IL-7 (PeproTech) for 2 days and then washed twice before being differentiated under T_H9-polarizing conditions.

Bi E., Ma X., Lu Y., Yang M., Wang Q., Xue G., Qian J., Wang S, & Yi Q. (2017). Foxo1 and Foxp1 play opposing roles in regulating the differentiation and antitumor activity of TH9 cells programmed by IL-7. Science signaling, 10(500), eaak9741.

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Intranasal IL-6 and Anti-IFN-γ in Pneumococcal Infection

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Mice were treated intranasally with 10 μg/30 μl/mouse recombinant mouse IL-6 protein (R&D) at the same time as the animals were challenged with S. pneumoniae. Mice were treated intranasally with 50 μg/20 μl/mice of anti-IFN-γ mAb (eBioscience) twice, once 1 day prior to S. pneumoniae challenge and the second at the time of challenge.
For the in vitro assays, macrophages were treated with 20 ng/ml of recombinant mouse IL-6 protein (R&D).

Gou X., Yuan J., Wang H., Wang X., Xiao J., Chen J., Liu S., Yin Y, & Zhang X. (2020). IL-6 During Influenza-Streptococcus pneumoniae Co-Infected Pneumonia—A Protector. Frontiers in Immunology, 10, 3102.

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In Vitro Polarization of Murine CD4+ T Cell Subsets

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Naive CD4⁺CD44⁻CD62L⁺ T cells were resuspended at 1 × 10⁶ cells/mL in RPMI 1640 supplemented with 10% HI-FCS, 50 μM β-mercaptoethanol, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomyocin and stimulated with plate-bound 1 μg/mL anti-CD3ε mAb and 1 μg/mL anti-CD28 (37.51, BioLegend) at 200 μL/well in a 96-well plate. Cells were polarized as indicated using the following culture conditions: Th0, 5 μg/mL anti-IL-4 mAb (11B11, BioLegend) and 5 μg/mL anti-IFN-γ mAb (XMG1.2, BioLegend); Th1, 10 ng/mL murine IL-12 (PeproTech) and 5 μg/mL anti-IL-4 mAb; Th2, 10 ng/mL murine IL-4 (PeproTech) and 5 μg/mL anti-IFN-γ mAb; Th17, 5 μg/mL anti-IL-4 mAb, 5 μg/mL anti-IFN-γ mAb, 10 ng/mL human IL-23 (PeproTech), 1 ng/mL human TGF-β (PeproTech), and 5 ng/mL murine IL-6 (PeproTech); T_reg, 1 ng/mL human TGF-β, 5 μg/mL anti-IL-4 mAb, and 10 μg/mL anti-IFN-γ mAb. After 72 hr cells were analyzed by intracellular flow cytometry for CD4⁺ Th subset polarization.

Akama-Garren E.H., Miller P., Carroll T.M., Tellier M., Sutendra G., Buti L., Zaborowska J., Goldin R.D., Slee E., Szele F.G., Murphy S, & Lu X. (2023). Regulation of immunological tolerance by the p53-inhibitor iASPP. Cell Death & Disease, 14(2), 84.

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