Tissue samples were dissociated with the Tumor Dissociation Kit, human (Miltenyi Biotech, UK) in combination with the gentleMACS™ Octo Dissociator (Miltenyi Biotech, GmbH) to obtain a gentle and rapid generation of single-cell suspensions. Then, TILs were magnetically isolated with antihuman CD3 microbeads (Miltenyi Biotech, UK) using the AutoMACS Pro Separator (Miltenyi Biotech, GmbH) and analyzed by polychromatic flow cytometry. In detail, TILs from dissociated tissues were characterized for the expression of CD4, CD8, CD25, CD127, IFN-γ, IL-4, IL-17, IL-9, IL-22, and FoxP3 using intracellular cytokine staining. Briefly, TILs were cultured in RPMI 1640 culture medium (SERO-Med GmbH, Wien) supplemented with 10% FCS HyClone (Gibco Laboratories, Grand Island, NY, USA) and stimulated for 5 h using the Leukocyte Activation Cocktail with BD GolgiPlug™ (BD Pharmingen). Cells were stained for surface antigens and then fixed with 4% (v/v) paraformaldehyde and permeabilized with 0.5% saponin, followed by intracellular staining with anti-IL-4, anti-IL-17, anti-IL-22, anti-IL-9, and anti-IFN-γ mAbs (BD Biosciences). For the detection of peripheral Tregs, TILs were fixed and permeabilized using the BD Pharmingen Human FoxP3 Buffer Set (BD Biosciences). A minimum of 10,000 events were acquired.
Anti ifn γ mab
Manufactured by BD
Sourced in United States
Anti-IFN-γ mAb is a monoclonal antibody that specifically binds to interferon-gamma (IFN-γ), a cytokine involved in the immune response. This product can be used for the detection and quantification of IFN-γ in various research and analytical applications.
Automatically generated - may contain errors
Lab products found in correlation
Anti il 17, by BD (3 mentions)
Anti il 22, by BD (3 mentions)
Golgiplug, by BD (3 mentions)
Anti human cd3 microbeads, by Miltenyi Biotec (2 mentions)
Hyclone fcs, by Thermo Fisher Scientific (2 mentions)
Gentlemacs octo dissociator, by Miltenyi Biotec (2 mentions)
Anti il 9, by BD (2 mentions)
Pharmingen human foxp3 buffer set, by BD (2 mentions)
Anti il 4, by BD (2 mentions)
Automacs pro separator, by Miltenyi Biotec (2 mentions)
Interleukin 2 (il 2), by BD (2 mentions)
Monensin, by BD (2 mentions)
Cd28 cd49d mab, by BD (2 mentions)
Perm wash solution, by BD (2 mentions)
Brefeldin a, by BD (2 mentions)
Anti il 4 mab, by BD (2 mentions)
Recombinant murine il 2, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharide lps derived from e coli serotype 0111 b4, by Merck Group (1 mentions)
Human tgf β, by R&D Systems (1 mentions)
Brefeldin a bfa, by Merck Group (1 mentions)
14 protocols using anti ifn γ mab
1
Characterization of Tumor-Infiltrating Lymphocytes
2
In Vitro T Cell Differentiation
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Recombinant murine IL15 and human TGFβ were purchased from R&D systems (Minneapolis, MN, USA) and recombinant murine IL2 was purchased from Peprotech (Hamburg, Germany). For in vitro stimulation, rIL15, rTGFβ, and rIL2 were used at a concentration of 10 ng/ml. Lipopolysaccharide (LPS) derived from E. coli (serotype 0111:B4) was purchased from Sigma-Aldrich (St. Louis, MO, USA). For CD4+ T cell differentiation, anti-IFNγ mAbs (5 μg/ml; clone XMG1.2, BD Biosciences) were added at the concentrations indicated in figure legends. Isolated naive CD4+CD62L+ T cells (1 × 105 cells/well) were cultured with 96-well plate-bound anti-CD3ε (10 μg/ml) + anti-CD28 (1 μg/ml) mAbs in the presence of rTGFβ (10 ng/ml) and rIL2 (10 ng/ml) for 5 days. For CD8+ T cell differentiation, MLN NK1.1+CD8+ T cell-depleted CD8+ T cells (5 × 105 cells/ml) isolated from either CD1d KO or Yeti/CD1d KO mice were cultured with rIL15 (10 ng/ml) in 24-well plates for 5 or 10 days.
3
Tissue-Infiltrating Lymphocyte Characterization
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Tissue pieces were dissociated with the Tumor Dissociation Kit, human (Miltenyi Biotech, UK) in combination with the gentleMACS™ Octo Dissociator (Miltenyi Biotech, GmbH), to obtain a gentle and rapid generation of single-cell suspensions. Then, TILs were magnetically isolated with antihuman CD3 microbeads (Miltenyi Biotech, UK) using AutoMACS Pro Separator (Miltenyi Biotech, GmbH) and analyzed by polychromatic flow cytometry. In detail, TILs from dissociated tissues were characterized for the expression of CD4, CD8, CD25, CD127, IFN-γ, IL-4, IL-17, IL-9, IL-22, and FoxP3 using intracellular cytokine staining. Briefly, TILs were cultured in RPMI 1640 culture medium (SERO-Med GmbH, Wien) supplemented with 10% FCS HyClone (Gibco Laboratories, Grand Island, NY, USA) and stimulated for 5 h using the Leukocyte Activation Cocktail, with BD GolgiPlug™ (BD Pharmingen). Cells were stained for the surface antigens, then fixed with 4% (v/v) paraformaldehyde and permeabilized with 0.5% saponin, followed by intracellular staining with anti-IL-4, anti-IL-17, anti-IL-22, anti-IL-9 and anti-IFN-γ mAbs (BD Biosciences). For the detection of peripheral Tregs, TILs were fixed and permeabilized using the BD Pharmingen Human FoxP3 Buffer Set (BD Biosciences). A minimum of 10,000 events was acquired.
4
PBMC Cytokine Profile in PDAC
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PBMCs were isolated by density gradient centrifugation from heparinized venous blood of PDAC patients or healthy subjects. PBMC samples were characterized for their expression of IL-22, IL-17 and IFN-γ using intracellular cytokine staining, as described previously [23] (link). Briefly, fresh or thawed PB-MCs were cultured in IMDM (Iscove's modified Dulbecco's medium) (BioWhittaker) supplemented with 10 % FBS (Invitrogen) and stimulated for 5 h with PMA (50 ng/ml) and ionomycin (500 ng/ml) in the presence of BFA (brefeldin A) (10 μg/ml) (all from Sigma-Aldrich). Cells were first stained for the surface antigens CD4 (BD Biosciences), then fixed with 4 % (w/v) paraformaldehyde and permeabilized with 0.5 % saponin, followed by intracellular staining with anti-IL-22, anti-IL-17 and anti-IFN-γ mAbs (BD Biosciences).
5
Quantifying CD8+ T Cell and Anti-IDUA IgG Response
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CD8+ T cells were magnetically isolated from the spleen of experimental mice (Miltenyi Biotec, 130-104-075). 105 CD8+ T cells were plated in triplicate in ELISPOT plates (Millipore, Bedford, MA) pre-coated with anti-IFN-γ capture mAb (2.5 μg/mL; BD Pharmingen, R46A2) in the presence of IL-2 (50 U/mL; BD Pharmingen) and 105 irradiated (6,000 rad) untransduced or LV.IDUA-transduced autologous EL-4 cells. After 42 h of incubation at 37°C and 5% CO2, plates were washed and IFN-γ-producing cells were detected by biotin-conjugated anti-IFN-γ mAb (0.5 μg/mL; BD Pharmingen, XMG 1.2). Streptavidin-HRP conjugate (Roche) was added. Total splenocytes or total BM (0.35 × 106 cells/well) was plated in complete RPMI in triplicate in ELISPOT plates pre-coated with rhIDUA (2 μg/well). After 24 h of incubation at 37°C and 5% CO2, plates were washed and anti-IDUA IgG-secreting cells were detected with peroxidase-conjugated rabbit anti-mouse immunoglobulin (Sigma A2554). All plates were reacted with H2O2 and 3-Amino-9-ethylcarbazole (Sigma, A6926). Spots were counted by ImmunoSpot reader (Cellular Technology).
6
Cyclin D1 Peptide-Specific T Cell Response
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Cultured PBMCs were restimulated with individual cyclin D1 15-mer peptides for 2 h. Then, Golgi-plug (BD Pharmingen) was added to the cultures and followed by another 4-h culture. After a total 6 h of stimulation, cells were harvested, surface stained with CD4 and CD8 mAbs, then fixed and permeabilized with Cytofix/Cytoperm solution (BD). Finally, the cells were stained intracellularly with anti-IFN-γ mAb (BD Pharmingen). The cells were acquired on Canton II or LSRII flow cytometer (BD Bioscience, San Jose, CA) and analyzed using FlowJo software (Treestar, Ashland, OR). When cultured T cells were analyzed, IFN-DCs were first loaded with cyclin D1 15-mer peptides for 1 h and then used to stimulated T cells.
7
Characterization of Anti-Tumor CD8+ T Cells
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CD107a degranulation and IFN-γ producing CD3+CD8+ T cells were identified within SMM MP-CTL by flow cytometry. Briefly, SMM MP-CTL were stimulated with HLA-A2+ or HLA-A2- MM cell lines, K562 cells, K562-A*0201 cells pulsed with respective peptide or K562-A*0201 cells alone in the presence of CD107a anti-human mAb. SMM MP-CTL alone served as a negative control. After 1 hour incubation, CD28/CD49d mAb (BD), as well as protein transport inhibitors Brefeldin A and Monensin (BD), were added for an additional 5 hours. Cells were harvested, washed in FACS buffer, and incubated with mAbs specific to CD3, CD8, CCR7, CD45RO, CD69 and/or CD137 antigens. After surface staining, cells were washed, fixed/permeabilized, stained with anti-IFN-γ mAb (BD), washed with Perm/Wash solution (BD), fixed in 2% paraformaldehyde, and analyzed by flow cytometry.
8
T Cell Activation and Regulation
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The CD8+ T cells and CD4+ T cells were isolated from the spleens or lymph nodes of BALB/c and DUC18 Tg mice using CD8a (Ly‐2) MicroBeads and CD4 (L3T4) MicroBeads according to manufacturer’s protocol (Miltenyi Biotec). The purity of isolated cells was greater than 95%. CD8+ T cells were stimulated by Dynabeads Mouse T‐Activator CD3/CD28 (Invitrogen) at several bead‐to‐cell ratios in the absence or presence of syngeneic CD4+ T cells (CD8 / CD4 ratio = 0.5). In some experiments, recombinant human IL‐2 (provided Takeda Pharmaceutical), recombinant mouse IL‐21 (R&D Systems), and/or recombinant mouse IFN‐γ (R&D Systems) were added. In some experiments, Transwell system (BD Biosciences) was used to separate CD8+ T cells from CD4+ T cells in the absence or presence of neutralizing mAb to cytokines, such as anti‐IL‐2 mAb (S4B6) (BD Biosciences), anti‐IL‐21 polyclonal Ab (BD Biosciences), or anti‐IFN‐γ mAb (H22) (kindly provided Dr RD Schreiber, Washington University School of Medicine).
9
Cytokine Production by Activated T Cells
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The CD4+ T cells cultured for five days were stimulated using an immobilized anti-TCR-β mAb (3 µg/mL, H57–597, BioLegend) for 16 h, and the culture supernatants were recovered. The amount of cytokines in the recovered supernatants was determined with ELISA using the following antibodies and reagents: anti-IL-4 mAb (Cat#554387, BD Biosciences), biotin-anti-IL-4 mAb (Cat#554390, BD Biosciences), anti-IL-5 mAb (Cat#554393, BD Biosciences), biotin-anti-IL-5 mAb (Cat#554397, BD Biosciences), anti-IFN-γ mAb (Cat#551216, BD Biosciences), biotin-anti-IFN-γ mAb (Cat#554410, BD Biosciences), anti-IL-2 mAb (Cat#554424, BD Biosciences), biotin-anti-IL-2 mAb (Cat#554426, BD Biosciences), streptavidin horseradish peroxidase (Cat#434323, Invitrogen), and TMB peroxidase EIA substrate kit (Cat#1721066, BioRad). For IL-13, the mouse IL-13 Duoset ELISA (Cat#DY413, R&D systems) was used for detecting the levels of the IL-13 cytokine. All antibodies and reagents were used according to the manufacturer’s protocols.
10
Cytokine Profiling of Treg-Mediated Immune Regulation
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CD25-depleted PBMC from HC or MS patients were cultured in presence or absence of Treg from independent healthy donors (ratio 1:1) and stimulated with 0.5 µg/mL anti-CD3 mAb (clone OKT3, Bio X Cell, West Lebanon, NH, USA). Supernatants were collected 72 h after stimulation. Cytokines were measured by Cytometric Bead Array (BD Biosciences) following manufacturer’s instructions and analyzed by GraphPad Prism6 (Statcon, Witzenhausen, Germany). For intracellular cytokine staining, anti-IFN-γ mAb (BD Biosciences) was used. Spleen cells from humanized mice were restimulated with 1 µg/mL Ionomycin and 1 ng/mL PMA for 5 h, 4 h in the presence of Monensin (1.3 µM/mL). Afterwards, cells were fixed and permeabilized (perm/fix solution; BD Pharmingen) and stained for IFN-γ.
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