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Anti ifnγ

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Anti-IFNγ is a laboratory reagent used to detect and measure the levels of the cytokine interferon-gamma (IFNγ) in biological samples. It is commonly used in flow cytometry and other immunoassay applications to analyze the immune response and T-cell activation.

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Lab products found in correlation

Brefeldin a, by Thermo Fisher Scientific (3 mentions) Anti cd3 145 2c11, by Thermo Fisher Scientific (2 mentions) Permeabilization buffer, by Thermo Fisher Scientific (2 mentions) Anti cd28, by Thermo Fisher Scientific (2 mentions) Anti cd4, by Thermo Fisher Scientific (2 mentions) Murine il 2, by Thermo Fisher Scientific (2 mentions) Anti il 4, by Cytek Biosciences (2 mentions) Anti ki67, by Thermo Fisher Scientific (1 mentions) Anti cd8, by Thermo Fisher Scientific (1 mentions) Collagenase a, by Roche (1 mentions) Anti foxp3, by Thermo Fisher Scientific (1 mentions) Anti tcrβ, by BD (1 mentions) Anti b220, by BD (1 mentions) Dnaase 1, by Roche (1 mentions) Anti cd4, by Cytek Biosciences (1 mentions) Anti nkp46, by BD (1 mentions) Foxp3 transcription factor staining buffer set, by Cytek Biosciences (1 mentions) Lsrfortessa cell analyzer, by BD (1 mentions) Phorbol myristate acetate (pma), by Merck Group (1 mentions) Il 12, by Cytek Biosciences (1 mentions)

7 protocols using anti ifnγ

1

Analyzing T Cell Cytokine Production in Nlrc3 Knockout Mice

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Splenocytes from WT and Nlrc3−/− mice were treated with ACK lysis buffer at room temperature for 1 min to remove red blood cells. Splenocytes were washed, counted and plated at 2 × 105 cells per well in a 96-well plate coated with 1 µg/mL anti-CD3 (145-2C11, Affymetrix eBioscience) and 1 µg/mL anti-CD28 (16–0281, Affymetrix eBioscience). Cells were cultured at 37°C with and without 20 ng/ml murine IL-2 (212-12, Peprotech) for 4 days. Brefeldin A (00–4506, Affymetrix eBioscience) was added to the media for 3 h, followed by washing in PBS, and staining with anti-CD4 (14–0042–85, Affymetrix eBioscience) and anti-CD3 (145-2C11, Affymetrix eBioscience) antibodies on ice for 20 min. Stained cells were fixed in 1% paraformaldehyde for 30 min on ice and permeabilized using permeabilization buffer (00–8333-56, Affymetrix eBioscience) according to manufacturer’s instructions. To detect intracellular cytokines, fixed cells were stained with anti-IFN-γ (50–7311, Tonbo) and anti-TNF (506322, Biolegend) for 30 min on ice. Flow cytometry were performed as described above.
Karki R., Man S.M., Malireddi R.K., Kesavardhana S., Zhu Q., Burton A.R., Sharma B.R., Qi X., Pelletier S., Vogel P., Rosenstiel P, & Kanneganti T.D. (2016). NLRC3 is an inhibitory sensor of PI3K–mTOR pathways in cancer. Nature, 540(7634), 583-587.
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2

Immunophenotyping of Ex Vivo Tumor Tissue

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For immunophenotyping of ex vivo tumor tissue, tumors were extracted on day 8, after bacteria treatment on days 0, 4, and 7. Lymphocytes were isolated from tumor tissue by mechanical homogenization and digestion with collagenase A (1 mg/ml, Roche) and DNAase I (0.5 μg/ml, Roche) in isolation buffer (RPMI 1640 supplemented with 5% FBS, 1% L-glutamine, 1% penicillin/streptomycin, and 10 mM Hepes) for 1 h at 37°C. Cells were then filtered through 100 μm cell strainers and washed in isolation buffer before staining. A Ghost Dye cell viability stain was used as a live/dead marker. Extracellular antibodies used include: anti-B220 (BD), anti-CD4 (Tonbo), anti-CD8 (eBioscience), and anti-NKp46(BD). Cells were then fixed using FOXP3/transcription factor staining buffer set (Tonbo) in accordance with the manufacturer’s protocol and then stained intracellularly. To measure the production of cytokines by T cells, cells were stimulated for 2 h with PMA (50 ng/ml; Sigma Aldrich) and ionomycin (1 nM; Calbiochem) in the presence of brefeldin A. We stained for intracellular markers using the following antibodies: anti-TCRβ (BD), anti-Ki67(Thermo), anti-TNF (eBioscience), anti-IFNγ (Tonbo), and anti-FOXP3 (eBioscience). Samples were analyzed using a BD LSR Fortessa cell analyzer. FlowJo was used for all data analyses.
Gurbatri C.R., Lia I., Vincent R., Coker C., Castro S., Treuting P.M., Hinchliffe T.E., Arpaia N, & Danino T. (2020). Engineered Probiotics for Local Tumor Delivery of Checkpoint Blockade Nanobodies. Science translational medicine, 12(530), eaax0876.
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3

Analyzing T Cell Cytokine Production in Nlrc3 Knockout Mice

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Splenocytes from WT and Nlrc3−/− mice were treated with ACK lysis buffer at room temperature for 1 min to remove red blood cells. Splenocytes were washed, counted and plated at 2 × 105 cells per well in a 96-well plate coated with 1 µg/mL anti-CD3 (145-2C11, Affymetrix eBioscience) and 1 µg/mL anti-CD28 (16–0281, Affymetrix eBioscience). Cells were cultured at 37°C with and without 20 ng/ml murine IL-2 (212-12, Peprotech) for 4 days. Brefeldin A (00–4506, Affymetrix eBioscience) was added to the media for 3 h, followed by washing in PBS, and staining with anti-CD4 (14–0042–85, Affymetrix eBioscience) and anti-CD3 (145-2C11, Affymetrix eBioscience) antibodies on ice for 20 min. Stained cells were fixed in 1% paraformaldehyde for 30 min on ice and permeabilized using permeabilization buffer (00–8333-56, Affymetrix eBioscience) according to manufacturer’s instructions. To detect intracellular cytokines, fixed cells were stained with anti-IFN-γ (50–7311, Tonbo) and anti-TNF (506322, Biolegend) for 30 min on ice. Flow cytometry were performed as described above.
Karki R., Man S.M., Malireddi R.K., Kesavardhana S., Zhu Q., Burton A.R., Sharma B.R., Qi X., Pelletier S., Vogel P., Rosenstiel P, & Kanneganti T.D. (2016). NLRC3 is an inhibitory sensor of PI3K–mTOR pathways in cancer. Nature, 540(7634), 583-587.
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4

T-Cell Differentiation Experiments

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T-cell differentiation experiments were performed as described previously.E8 Briefly, FoxP3- CD4+ T cells were purified and activated using anti-CD3/CD28 beads (Dynabeads, Invitrogen). For TH1 differentiation, 1 × 105 T cells were stimulated in the presence of IL-2 (20 U/mL; eBioscience), IL-12 (20 ng/mL; Tonbo), and anti–IL-4 (10 μg/mL; Tonbo). For Treg-cell differentiation, cells were stimulated in the presence of IL-2 (100 U/mL; eBioscience), TGF-β (5 ng/mL; Tonbo), and anti–IFN-γ (10 μg/mL; Tonbo). After 4 days, T cells were stimulated with ionomycin (500 ng/mL) and phorbol 12-myristate 13-acetate (50 ng/mL) in the presence of monensin (eBioscience) for 4 hours before intracellular staining. FoxP3 staining was performed using FoxP3/transcription factor staining kit (Tonbo).
Ku M., Ke E., Sabouri-Ghomi M., Abadejos J.R., Freeman B., Nham A., Phillips N., Yang K.Y., Lui K.O, & Kirak O. (2018). Deconstructive somatic cell nuclear transfer reveals novel regulatory T-cell subsets. The Journal of allergy and clinical immunology, 142(3), 997-1000.e4.
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5

Isolation and Characterization of Intestinal Immune Cells

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Single-cell suspensions were prepared from the colon as previously described.35 The following monoclonal antibodies were used to stain and sort cells- Epcam (clone G8.8), CD45.2 (clone 104), CD90.2 (clone 30-H12), and MHCII (clone M5/114.152). Splenic and MLN cells were processed and stimulated as previously described.35 Briefly, single-cell suspensions were prepared and stimulated with phorbol-12-myristate-13-acetate and ionomycin for 4 h, with brefeldin A (00-4506, Affymetrix eBioscience) added for the last 2 h. For intracellular cytokine staining, cells were fixed and permeabilized by using a fixation and permeabilization solution (00-5523, eBioscience). Antibodies used for staining were anti-CD3 (clone 145 – 2C11), anti-CD4 (clone RM4-5), anti-CD19 (clone 1D3), anti-IFNγ (50-7311, Tonbo), and anti- TNF (506322, Biolegend). Flow cytometry data were acquired on an LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (TreeStar).
Sharma D., Malik A., Guy C.S., Karki R., Vogel P, & Kanneganti T.D. (2017). Pyrin Inflammasome Regulates Tight Junction Integrity to Restrict Colitis and Tumorigenesis. Gastroenterology, 154(4), 948-964.e8.
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6

Isolation and Culture of Naive T Cell Subsets

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T cells were enriched from spleens and LNs using the MagniSort CD4 negative selection kit (Thermo Fisher Scientific). Naive CD4+ T cells were isolated by flow cytometry on the basis of markers CD4+CD62L+CD44−CD25− or using the EasySep mouse naive T cell isolation kit. 105 naive T cells were cultured for 4 d (Th17 and induced T reg [iT reg] cells) in a 96-well flat-bottom plate coated with 2 µg/ml anti-CD3 (clone 2C11; Tonbo Biosciences) and 2 µg/ml anti-CD28 (clone 37.51; Tonbo Biosciences) with the relevant cytokines and blocking antibodies: classical Th17 (20 ng/ml IL-6, 2 ng/ml TGFβ, 10 µg/ml anti-IL4 [clone 11B11; Tonbo Biosciences], and 10 µg/ml anti-IFNγ [clone XMG1.2; Tonbo Biosciences]), pathogenic Th17 (20 ng/ml IL-6, 20 ng/ml IL-1β, 20 ng/ml IL-23, 10 µg/ml anti-IL4, and 10 µg/ml anti-IFNγ), iT reg cells (20 ng/ml TGFβ and 100 U/ml IL-2), or Th0 cells (100 U/ml IL-2). Th17 cell cultures were performed in Iscove’s medium, and iT reg cell cultures were performed in RPMI medium. All media were supplemented with 10% FBS, penicillin/streptomycin, glucose, pyruvate, β-mercaptoethanol, and Hepes. Cytokines were purchased from R&D Systems (murine IL-6 and human IL-2), Miltenyi Biotec (murine IL-1β and murine IL-23), or ProteinTech (HumanKine; human TGFβ).
Warshauer J.T., Belk J.A., Chan A.Y., Wang J., Gupta A.R., Shi Q., Skartsis N., Peng Y., Phipps J.D., Acenas D., Smith J.A., Tamaki S.J., Tang Q., Gardner J.M., Satpathy A.T, & Anderson M.S. (2021). A human mutation in STAT3 promotes type 1 diabetes through a defect in CD8+ T cell tolerance. The Journal of Experimental Medicine, 218(8), e20210759.
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7

Multiparameter Flow Cytometry Protocol

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Sertraline, Haloperidol, Paroxetine, Fluvoxamine, and EDTA were purchased from Sigma-Aldrich (St Louis, USA). The following regents were purchased from BD Biosciences (Auckland, New Zealand): A5-FITC, -PE, -APC, A5 binding buffer, mouse CD4-APC Cy7, -PE Cy7, anti-mouse CD3-APC, anti-mouse CD45.2 PerCP Cy5.5, anti-mouse Ly6G-PE, TNFα-FITC, RPMI 1640 medium, penicillin, and streptomycin. Anti-mouse CD8a-e450, anti-mouse CD11b-PE Cy7, TO-PRO-3 iodide, PO-PRO-1, hoechst 33342, NP-FITC, Live Dead-APC, -Yellow, and foetal calf serum (FCS) were purchased from Thermofisher (Scoresby, Australia). Anti-mouse CD14-PerCP Cy5.5, anti-mouse CD45.2-FITC, anti-mouse CD8-PE, and anti-mouse CD11c-APC Cy7 were purchased form Biolegend (San Diego, USA). HA-FITC was purchased from Santa Cruz Biotehnology (Texas, USA), and NP-FITC was purchased from Invitrogen (Carlsbad, USA). Anti-IFN-γ was purchased from Tonbo Biosciences (Ferris Square, USA), and MycoZap reagent was purchased from Lonza (Basel, Switzerland). Filcol Paque Premium was purchased from GE Healthcare Life Science (Chicago, USA).
Atkin-Smith G.K., Duan M., Zanker D.J., Loh L., Nguyen T.H., Koutsakos M., Nguyen T., Jiang X., Carrera J., Phan T.K., Liu C., Paone S., Oveissi S., Hodge A.L., Baxter A.A., Kedzierska K., Mackenzie J.M., Hulett M.D., Bilsel P., Chen W, & Poon I.K. (2020). Monocyte apoptotic bodies are vehicles for influenza A virus propagation. Communications Biology, 3, 223.
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