Caco-2 cells, obtained from ATCC, were grown in high glucose Dulbecco’s modified Eagle’s media (DMEM) supplemented with 10% FBS, 1% l -glutamine and 1% penicillin/streptomycin. Cells were maintained at 37°C under a humidified atmosphere of 5% CO2 in air. Experimental inflammatory condition in Caco-2 cell monolayers was induced by exposure for different times according to the assays, to 10 ng/mL recombinant human IFN-γ (Sigma-Aldrich) for 3 h and then 10 ng/mL TNF-α (Sigma-Aldrich) or to 500 μM H2O2, as previously described (Catanzaro et al., 2015 (link)). A 24 h pre-treatment with CBD, CBDA, THC and THCA (0.01–10 μg/mL) was applied before the stimulation. Reagents for cell cultures were from Cambrex-Lonza (Basel, Switzerland) and FBS from Gibco, Invitrogen (Carlsbad, CA).
Recombinant human ifn γ
Manufactured by Merck Group
Sourced in United States
Recombinant human IFN-γ is a laboratory-produced protein that is structurally and functionally similar to the natural human interferon-gamma (IFN-γ) cytokine. It is used in research applications to study the biological functions and properties of IFN-γ.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Mccoy s 5a, by American Type Culture Collection (2 mentions)
Mccoy s 5a, by Thermo Fisher Scientific (2 mentions)
Incb024360, by Selleck Chemicals (2 mentions)
Incb024360, by Merck Group (2 mentions)
Epacadostat, by Selleck Chemicals (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Tnf α, by Merck Group (1 mentions)
Ficoll gradient, by GE Healthcare (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Phorbol 12 myristate 13 acetate, by Merck Group (1 mentions)
Edta free protease inhibitor cocktail, by Roche (1 mentions)
Recombinant human m csf, by Merck Group (1 mentions)
Recombinant human il 4, by Merck Group (1 mentions)
Cd14 percp cy5, by BioLegend (1 mentions)
14 protocols using recombinant human ifn γ
1
Caco-2 Inflammatory Model Analysis
2
Priming Mesenchymal Stem Cells with IFN-γ
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To generate IFN-γ pre-stimulated (primed) BMMSC and AdMSC, cells were seeded at the same density in all media formulations supplemented with 50 ng/mL of recombinant human IFN-γ (Sigma). IFN-γ treatment lasted three days as previously described14 (link). All experiments were performed in comparison to vehicle- (1× phosphate buffer solution, PBS, supplemented with 0.1% bovine serum albumin, BSA) treated cells.
3
Macrophage Derivation from Healthy Donors
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All procedures for macrophage derivation were approved by local Ethical Committee (Hospital das Clínicas/UFG, prot. 132/2012). Venous blood samples (~12 mL) were obtained from healthy blood donors (Blood Bank of Instituto Goiano de Hematologia e Oncologia, Goiânia, Goiás, Brazil) in EDTA-vacuum tubes (Greiner bio-one, Vacuette, Americana, SP, Brazil). Blood was layered on Ficoll gradient (GE Healthcare Bio-Sciences AB, Uppsala, Uppsala län, Sweden) and centrifuged at 1,000 g, r.t., 20 min. Mononuclear cells (5 × 105 in 500 μL RPMI 1640 medium supplemented with 10% FCS, 11 mM sodium bicarbonate, 2 mM L-glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (all reagents from Sigma)), were distributed onto 13 mm-round glass coverslips (Knitel, Varrentrappstr, Bielefeld, Germany) and placed in 24-well plates (TPP, Techno Plastic Products, Trasadingen, Swizerland). Culture medium was replaced each 48 h during 7 days (36°C, 5% CO2). Nonadherent cells were removed during medium changes and adherent monocytes differentiated into macrophages. For some experiments, at the 6th day of culture cells were treated with 10 ng/mL of recombinant human IFNγ (Sigma) overnight, before macrophage infection.
4
Inhibitors Modulate Kynurenine in Tumor Cells
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SKOV3 human ovary adenocarcinoma (IDO1+) and A172 human glioblastoma (TDO+) cell lines were obtained from ATCC and cultured in McCoy’s 5A (10% FBS) and Dulbecco’s Modified Eagle’s Medium (DMEM, 10% FBS) culture media, respectively. Cell lines were incubated at 37°C in 96 well plates with either the IDO1 inhibitor INCB024360 (Epacadostat, Selleck) at 2 μM for SKOV3 cells and 20 μM for A172 cells; the TDO inhibitor 680C91 (WuXi Apptec) at 20 μM for all cells; or Kynureninase (Mp-KYNase) at 0.36 mg/ml for all cells. In addition, A172 cells were stimulated with recombinant human IFNγ (100 ng/mL, Sigma) to induce IDO1 expression to determine the effects on Kynureninase, INCB024360 and 680C91 on Kyn levels made by IDO1/TDO dual expressing cells. After 48 h, cell supernatants were collected, snap frozen in liquid nitrogen and stored at −80°C. Kynurenine concentrations of the supernatant were then determined by LC/MS. McCoy’s 5A, DMEM and FBS were all purchased from GIBCO®.
5
Immune Cell Stimulation Reagents
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EDTA-free protease inhibitor cocktail was purchased from Roche Applied Science (Indianapolis, IN). Other reagents such as recombinant human IFN-γ (Minneapolis, MN), lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) from Sigma-Aldrich (St. Louis, MO), synthetic double-stranded DNA [Poly(dA:dT)] in complex with transfection reagent (LyoVec) and nigericin from InvivoGen (San Diego, CA), and Bay11-782 were purchased from Sigma-Aldrich.
6
Inhibitors Modulate Kynurenine in Tumor Cells
7
Monocyte Differentiation and Activation
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Cytokines were purchased from Peprotech (Cranbury, NJ, USA): recombinant human IL-4 (#200-04), recombinant human IL-13 (#200-13), recombinant human IL-10 (#200-10), recombinant human M-CSF (#300-25), recombinant human GM-CSF (#300-03), recombinant human IFN-γ (#300-02) and Sigma-Aldrich (St. Louis, MO, USA): LPS (#L3024). Mouse anti-human antibodies were purchased from BD Biosciences (Franklin Lakes, NJ, USA CD11c FITC (#561355), CD11b FITC (#562793), CD40 BV711 (#563397), CD40 BV510 (#563456), CD80 BV480 (#562432), CD86 BV421 (#562432), MHC-II BV605 (#740408) and from Biolegend (San Diego, CA, USA): CD163 APC (#333610), CD14 PerCp Cy5.5 (#325622), CD206 PE (#321106).
8
Infection of B-LCLs with S. Typhi
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B-LCLs were incubated in RPMI in a 37°C 5% CO2 incubator with wild-type S. Typhi strain Ty2 at different cell:bacteria multiplicity of infection (MOI) (18 (link)) for 3 hours. Cells left uninfected were used as controls. S. Typhi strain Ty2 was obtained through the cryobank at the Center Vaccine Development and Global Health (CVD). After 16-18 hours of gentamicin treatment, B-LCLs were irradiated (6,000 rads), and surface stained with a monoclonal antibody (mAb) to CD45, a marker abundantly expressed on the surface of hematopoietic cells (46 (link)). After staining, autologous B-LCL were co-cultured with overnight-rested PBMC at a B-LCL : PBMC cell ratio of 1:5 in R10 media (i.e., RPMI 1640 (Gibco, Grand Island, New York) media supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 50 µg/mL gentamicin, 2 mM L-glutamine, 1 mM sodium pyruvate, 10 mM HEPES (Gibco) buffer and 10% heat-inactivated fetal bovine serum). When noted, human recombinant IFN-γ (Sigma St. Louis, MO, USA) was added to the co-culture at 0.5, 5, or 25 ng/ml, Time-course experiments were used to track phenotypic changes (3, 6, and 16 hours). Subsequently, cells were labeled and evaluated by conventional flow cytometry or mass cytometry as described below.
9
Characterization of Anthocyanin-Rich Bilberry Extract
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All reagents were of analytical grade and obtained commercially. Monoclonal rabbit anti-human phospho-STAT1 (Tyr701; D4A7), polyclonal rabbit anti-human STAT1 were obtained from Cell Signaling Technologies (Danvers, MA, United States).
Human recombinant IFN-γ was obtained from Sigma (Sigma-Aldrich, St. Louis, MO, United States). The anthocyanin-rich bilberry extract (ARBE) was manufactured by Kaden Biochemicals, Symrise GmbH & Co (Holzminden, Germany) and was allocated as a powder (25% anthocyanin content). This powder was dissolved in ddH2O to establish a stock suspension of 10 mg ARBE/ml. Due to sedimentation, the stock suspension was homogenized by strong agitation during 30 minutes prior to each usage. A detailed analysis of the ARBE powder can be found inS1 File .
Human recombinant IFN-γ was obtained from Sigma (Sigma-Aldrich, St. Louis, MO, United States). The anthocyanin-rich bilberry extract (ARBE) was manufactured by Kaden Biochemicals, Symrise GmbH & Co (Holzminden, Germany) and was allocated as a powder (25% anthocyanin content). This powder was dissolved in ddH2O to establish a stock suspension of 10 mg ARBE/ml. Due to sedimentation, the stock suspension was homogenized by strong agitation during 30 minutes prior to each usage. A detailed analysis of the ARBE powder can be found in
10
Glioma Cell Stimulation Assay
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The U87-MG (GBM of unknown origin) and U118-MG (GBM of unknown origin) cell lines were obtained from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. The cell lines were authenticated via STR profiling. Cells were cultured in high-glucose DMEM (cat. no. 04-052-1ACS; Biological Industries), containing 10% FBS (cat. no. 04-001-1ACS; Biological Industries) and 1% penicillin-streptomycin (cat. no. P1400; Beijing Solarbio Science & Technology Co., Ltd.) at 37°C in a humidified incubator with 5% CO2.
Reagents included lipopolysaccharide (LPS; 4 µg/ml; cat. no. L7770; Sigma-Aldrich; Merck KGaA) and human recombinant IFN-γ (40 ng/ml; cat. no. 11725-HNAS; Sino Biological, Inc.). Glioma cells were stimulated with LPS and IFN-γ at 37°C in a humidified incubator with 5% CO2 for 0, 4, 8 or 24 h.
Reagents included lipopolysaccharide (LPS; 4 µg/ml; cat. no. L7770; Sigma-Aldrich; Merck KGaA) and human recombinant IFN-γ (40 ng/ml; cat. no. 11725-HNAS; Sino Biological, Inc.). Glioma cells were stimulated with LPS and IFN-γ at 37°C in a humidified incubator with 5% CO2 for 0, 4, 8 or 24 h.
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