NK cells from healthy donors were cultured in RPMI-1640 plus 10% FBS in 48-well flat-bottom microtiter plates (5 × 104 cells per well) in the absence or presence of fibroblasts (NK:fibroblast ratio, 2.5:1), and 100 IU/mL rhIL-2 (R&D systems, Oxford, United Kingdom) was added when indicated. At the indicated time intervals, NK cells were harvested, counted and analyzed. When indicated, 0.5 mM 1-methyl-tryptophan (Sigma, St. Louis, MO, USA) and/or 5 μM NS398 (Cayman Chemicals, Ann Arbor, MI, USA) were added at the onset of the co-cultures.
1 methyl tryptophan
Manufactured by Merck Group
Sourced in United States
1-methyl-tryptophan is a chemical compound that is commonly used as a laboratory reagent. It is a tryptophan derivative with a methyl group attached to the nitrogen atom. This compound is often utilized in various biochemical and cell biology research applications, but its core function is to serve as a tool for investigating specific biological processes.
Automatically generated - may contain errors
4 protocols using 1 methyl tryptophan
1
NK Cell Activation Assay with Fibroblasts
2
Coculture of NK cells and NB cells
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For coculture experiments freshly isolated NK cells were cultured (3×105 cells/well) with 600 U/mL of IL-2 either alone or in the presence of NB cell lines (105 cells/well) in 24-wells plates in a 3:1 ratio. For transwell culture conditions, we used insert for 24-well plate with Polyester (PET) membrane bottom and pore size 0.4 µm (Sarstedt, Nümbrecht, Germany). Freshly isolated NK cells were seeded in the upper insert (3×105 cells/well) and both NB (105 cells/well) and NK cells were seeded in the lower chamber (3×105 cells/well) using a 3:1 ratio.
In the experiments with indoleamine 2,3-dioxygenase (IDO) and Prostaglandin E2 (PGE2) synthesis inhibitors, in addition to 600 U/mL of IL-2 for 4 days, as previously described,25 (link) we used: 1 mM of 1-methyl-tryptophan (Sigma-Aldrich, an IDO inhibitor capable to block kynurenine production) and 5 µM NS-398 (Sigma-Aldrich, an inhibitor of PGE2 synthesis). NK cells were collected and used as effectors in the cytotoxicity assays. K-562 cell line was used as control target for NK cell functional assays.
In the experiments with indoleamine 2,3-dioxygenase (IDO) and Prostaglandin E2 (PGE2) synthesis inhibitors, in addition to 600 U/mL of IL-2 for 4 days, as previously described,25 (link) we used: 1 mM of 1-methyl-tryptophan (Sigma-Aldrich, an IDO inhibitor capable to block kynurenine production) and 5 µM NS-398 (Sigma-Aldrich, an inhibitor of PGE2 synthesis). NK cells were collected and used as effectors in the cytotoxicity assays. K-562 cell line was used as control target for NK cell functional assays.
3
Inhibition of Indoleamine 2,3-Dioxygenase in HPV Tumors
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Indoleamine 2,3 dioxygenase inhibition was achieved by chronic administration of
either a nonspecific competitive inhibitor of the enzyme, 1-methyl tryptophan
(Sigma-Aldrich, Saint-Louis, Missouri, USA; catalog numbers 447439 and 860646),
or a selective IDO1 inhibitor30 (link) (hereafter referred to as IDOInh; kindly provided by Boehringer
Ingelheim, Ingelheim, Germany). The levogyre and racemic forms of 1-methyl
tryptophan (L-1MT and DL-1MT, respectively) were used in 2 different
experiments. Both forms inhibit IDO1 but they are also active against
IDO231 (link),32 (link) and TDO, although this last effect is exerted in a
noncompetitive manner.33 (link) 1L-MT and 1-DL-MT were given in the drinking water starting on day 2 and
ending on day 30 after implantation of HPV-positive tumor cells. 1L-MT was
administered at a dose of 5 mg/mL, whereas 1-DL-MT was administered at a dose of
2 mg/kg. Due to poor solubility, 1-MT was first dissolved in NaOH and then
diluted to concentration and titrated to a pH of 10 using HCl; the solution was
freshly made every other day. The pharmacokinetic properties of IDOInh and its
ability to selectively block IDO1 have been previously described.30 (link),34 (link) IDOInh was
administered by oral gavage at the daily dose of 200 mg/kg, starting on day 10
and ending on day 28 after implantation of HPV-positive tumor cells.
either a nonspecific competitive inhibitor of the enzyme, 1-methyl tryptophan
(Sigma-Aldrich, Saint-Louis, Missouri, USA; catalog numbers 447439 and 860646),
or a selective IDO1 inhibitor30 (link) (hereafter referred to as IDOInh; kindly provided by Boehringer
Ingelheim, Ingelheim, Germany). The levogyre and racemic forms of 1-methyl
tryptophan (L-1MT and DL-1MT, respectively) were used in 2 different
experiments. Both forms inhibit IDO1 but they are also active against
IDO231 (link),32 (link) and TDO, although this last effect is exerted in a
noncompetitive manner.33 (link) 1L-MT and 1-DL-MT were given in the drinking water starting on day 2 and
ending on day 30 after implantation of HPV-positive tumor cells. 1L-MT was
administered at a dose of 5 mg/mL, whereas 1-DL-MT was administered at a dose of
2 mg/kg. Due to poor solubility, 1-MT was first dissolved in NaOH and then
diluted to concentration and titrated to a pH of 10 using HCl; the solution was
freshly made every other day. The pharmacokinetic properties of IDOInh and its
ability to selectively block IDO1 have been previously described.30 (link),34 (link) IDOInh was
administered by oral gavage at the daily dose of 200 mg/kg, starting on day 10
and ending on day 28 after implantation of HPV-positive tumor cells.
4
Termination Arrest Bypass Assay
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Individual colonies of the OSYRIS cells carrying the PTC library mutants were inoculated in the wells of a 96-well plate containing 120 µl of LB media, supplemented with 50 μg/ml Amp, 25 μg/ml Kan, 25 μg/ml Spc, and 15 μg/ml Chl, and grown for 24 h at 37 °C with constant shaking. Cultures were diluted 1:40 (v/v) in 120 µl of fresh LB supplemented with the same antibiotics, 0.35 mg/ml 1-methyl-tryptophan (Sigma) and 0.016 ng/ml of HSL. Plates were placed into TECAN Infinite M200 Pro plate reader and incubated at 37 °C with constant linear (3 mm) shaking. Optical density (A600) of the cultures and oGFP fluorescence were monitored as described above.
The termination arrest bypass score was calculated by comparing the efficiency of GFP expression in the OSYRIS cells carrying GFP-TnaC(W12R) mutant construct to that in the OSYRIS cells carrying wt GFP-TnaC construct. The SB score values were computed based on the readings obtained at the 48 h time point using the following formula: where RFU is relative fluorescence units.
The mean SB score values were calculated using data obtained in two independent experiments.
The termination arrest bypass score was calculated by comparing the efficiency of GFP expression in the OSYRIS cells carrying GFP-TnaC(W12R) mutant construct to that in the OSYRIS cells carrying wt GFP-TnaC construct. The SB score values were computed based on the readings obtained at the 48 h time point using the following formula: where RFU is relative fluorescence units.
The mean SB score values were calculated using data obtained in two independent experiments.
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