Purified CD4+ or CD8+ human T cells (105 cells) were stimulated with either 105 irradiated (9000 rad) allogeneic T-cell-depleted PBMCs (MLR – mixed lymphocytes reaction) plus 1 μg ml−1 of anti-CD3 mAb (BD Biosciences); or 5 μg ml−1 anti-CD3 plus 5 μg ml−1 anti-CD28 mAb (BD Biosciences), in the absence or presence of different numbers of tumour-MSCs (2 × 104,104,103,102 per well). Co-culture experiments were performed in RPMI 1640 medium (Seromed), supplemented as indicated in the first paragraph. On day 5, cultures were pulsed for 8 h with 0.5 μCi (0.0185 MBq) of 3H-TdR (Amersham, Little Chalfont, UK) harvested, and radionuclide uptake was measured by scintillation counting. Some experiments were conducted in the presence of the IDO1 inhibitor 1-methyl-L-tryptophan (1, 0.5, or 0.25 mM ) or in the presence of the COX inhibitor indomethacin (1, 0.5, 0.25 mcM ) purchased from Sigma Aldrich.
1 methyl l tryptophan
Manufactured by Merck Group
Sourced in United States
1-methyl-L-tryptophan is a chemical compound that serves as a laboratory reagent. It is derived from the amino acid tryptophan and has the molecular formula C₁₁H₁₂N₂O₂. The compound is used in various experimental and analytical applications, but a detailed description of its core function is not available while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
L kynurenine sulfate salt, by Merck Group (2 mentions)
L tryptophan, by Merck Group (2 mentions)
1 methyl d tryptophan, by Merck Group (2 mentions)
Recombinant human ifn γ, by Thermo Fisher Scientific (2 mentions)
Anti cd3 mab, by BD (1 mentions)
3h tdr, by Cytiva (1 mentions)
Anti cd28 mab, by BD (1 mentions)
Indomethacin, by Merck Group (1 mentions)
Anti cd3, by BD (1 mentions)
Polyethylene glycol 400 peg400, by Sinopharm (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Dmem medium, by Thermo Fisher Scientific (1 mentions)
Ova b16, by National Collection of Authenticated Cell Cultures (1 mentions)
Modulus 2 microplate multimode reader, by Promega (1 mentions)
Nano glo luciferase assay system, by Promega (1 mentions)
Ecl detection system, by Cytiva (1 mentions)
Primary rabbit anti human ido1, by Merck Group (1 mentions)
Secondary horseradish peroxide coupled goat anti rabbit igg h l, by Fortis Life Sciences (1 mentions)
1 methyl dl tryptophan, by Merck Group (1 mentions)
Sodium dithionate, by Merck Group (1 mentions)
9 protocols using 1 methyl l tryptophan
1
Immune Cell Suppression by Tumor-Derived MSCs
2
In Vitro Cultivation of Tumor Cell Lines
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Mouse tumor cell lines B16 (melanoma), OVA-B16 (melanoma), C26 (colon cancer) and 4 T1 (breast cancer) were purchased from the China Center for Type Culture Collection (Beijing, China) and cultured in RPMI 1640 (Thermo Fisher, MA, USA) with 10% fetal bovine serum (FBS) (Gibco, MA, USA), with the exception of 4 T1 cells, which were grown in DMEM medium (Gibco, MA, USA) with 10% FBS.
Carboxyamidotriazole was synthesized by the Institute of Materia Medica, Chinese Academy of Medical Sciences (Beijing, China). Polyethylene glycol 400 (PEG400) was obtained from Sinopharm Chemical Reagent Beijing (Beijing, China). 1-Methyl-L-tryptophan, 3′, 4′-dimethoxyflavone and L-kynurenine sulfate salt were purchased from Sigma-Aldrich (Saint Louis, USA).
Carboxyamidotriazole was synthesized by the Institute of Materia Medica, Chinese Academy of Medical Sciences (Beijing, China). Polyethylene glycol 400 (PEG400) was obtained from Sinopharm Chemical Reagent Beijing (Beijing, China). 1-Methyl-L-tryptophan, 3′, 4′-dimethoxyflavone and L-kynurenine sulfate salt were purchased from Sigma-Aldrich (Saint Louis, USA).
3
Modulation of HIV-1 Infection in Macrophages
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Macrophages were plated in one well of a 96-well plate, treated with SIV3 +or SIV3+ ΔVpx virus like particles for 10 hr, followed by treatment with 0.3% DMSO, 0.5 μM FICZ, 3 μM CH-223191 or 1000 U/ml of IFN-γ; or indicated amount of L-Tryptophan and 1-Methyl-L-Tryptophan (Sigma) for 16 hr before infection with HIV-1NLYU2 nluc. Cells were lysed 48 hr post infection and luciferase was measured using the Nano-Glo Luciferase Assay System (Promega) and Modulus II Microplate Multimode Reader (Turner BioSystems).
4
Quantification of IDO1 and AhR in MSCs
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Approximately 1 million MSCs were harvested from a single 75-cm2 flask at 80% confluency. Cells had been treated for 12h with 50 ng/ml recombinant human IFN-γ (Invitrogen, Carlsbad, CA), and/or 1-methyl-DL-tryptophan, 1-methyl-D-tryptophan, or 1-methyl-L-tryptophan (Sigma-Aldrich, St. Louis, MO). Whole-cell protein lysates were run in a 4-20% polyacrylamide gel electrophoresis apparatus and then transferred to PVDF membrane, which was blocked in 5% non-fat milk in Tris-buffered saline + 0.05% Tween-20. Protein was detected using primary rabbit anti-human IDO1 (1:1000; EMD Millipore Corporation, Billerica, MA), primary mouse anti-human AHR (1:1000; ThermoFisher, Waltham, MA) or primary rabbit anti-human β-actin (1:1000; Cell Signaling Technology, Danvers, MA), and secondary horseradish peroxide-coupled goat anti-rabbit IgG h + l (1:10,000; Bethyl Laboratories, Montgomery, TX). ECL detection system (Amersham Pharmacia Biotech, Piscataway, NJ) was used to detect immunoreactive blots.
5
Formulation and Administration of 1-MT in MYR
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We purchased 1-methyl-L-tryptophan (purity 95%) from Sigma-Aldrich (Deisenhofen, Germany)
and used the triglyceride Myritol®318 (MYR) from Caesar und Loretz GmbH
(Hilden, Germany) as an excipient. MYR was filtered using sterile 0.2 μm
polytetrafluoroethylene filters (ReZist, Whatman GmbH, Dassel, Germany) prior to its
addition to the separately supplied 1-MT. Pre-weighed 1-MT was provided in injection
vials, and suspensions were prepared immediately prior to administration. MYR was added to
the 1-MT via injection through the septum of the vial, followed by careful mixing and
withdrawal of 4.2 ml of the suspension (corresponding to 0.5 g of 1-MT and 4.0 ml of MYR).
The suspension was again gently mixed immediately before s.c. application to avoid
sedimentation of 1-MT.
and used the triglyceride Myritol®318 (MYR) from Caesar und Loretz GmbH
(Hilden, Germany) as an excipient. MYR was filtered using sterile 0.2 μm
polytetrafluoroethylene filters (ReZist, Whatman GmbH, Dassel, Germany) prior to its
addition to the separately supplied 1-MT. Pre-weighed 1-MT was provided in injection
vials, and suspensions were prepared immediately prior to administration. MYR was added to
the 1-MT via injection through the septum of the vial, followed by careful mixing and
withdrawal of 4.2 ml of the suspension (corresponding to 0.5 g of 1-MT and 4.0 ml of MYR).
The suspension was again gently mixed immediately before s.c. application to avoid
sedimentation of 1-MT.
6
Enzyme Culture Material Protocol
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The enzyme culture materials Potassium phosphate, dipotassium hydrogen phosphate, 1-methyl-l -tryptophan, l -kynurenine, l -tryptophan, catalase, trichloroacetic acid (TCA), p-Dimethylaminobenzaldehyde (Ehrlich’s reagent) and sodium dithionate were purchased from sigma Aldrich and methylene blue, dimethyl sulfoxide (DMSO), glacial acetic acid, ascorbic acid, glycerol and tris-HCL buffer were purchased from Sisco Research Laboratories, India. The recombinant human IDO/TDO protein were purchased from Biovision (Catalog no.: P1096 human recombinant IDO1 and CSB-YP023351HU human recombinant TDO2). All the other HPLC grade solvent were purchased from Merck Pvt. Ltd. The IDO/TDO assays were performed according to the reported procedures.1 and 2, the details of which is mentioned in the SI.
7
Evaluating Compounds' Effects on CYP1A1
8
Kynurenine Production in IFN-γ-Treated Cells
9
Synthesis and Procurement of Compounds
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Carboxyamidotriazole was synthesized by the Institute of Materia Medica, Chinese Academy of Medical Sciences (Beijing, China).Oxaliplatin, Irinotecan and Paclitaxel were purchased from Selleck Chemicals (Los Angeles, USA). 1-Methyl-L-tryptophan, 3′, 4′-Dimethoxy avone and L-Kynurenine sulfate salt were purchased from Sigma-Aldrich (Saint Louis, USA).
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