Sulfosalicylic acid solution

Tryptophan, ornithine, and arginine were measured by high-pressure liquid chromatography (HPLC) as indicators of IDO and arginase-1 activity. After a 5-day TIDC and T-cell coculture, the supernatant was collected and tryptophan, ornithine, and arginine concentrations were measured. In brief, 200 μL of cell supernatant was subjected to a deproteinization step using a 30% sulfosalicylic acid solution (Sigma). Then, supernatants were diluted with Jeol sampling buffer (JEOL) containing 0.2 μmol/mL of aminoethyl cysteine and glucosaminic acid (internal standards) (Sigma). Supernatants (50 μL) were then injected into an automated amino acid analyzer (JEOL Aminotac 500) and eluted with lithium citrate buffer. Tryptophan, ornithine, and arginine were detected at 570 nm. Data acquisition and calculations were made using the JEOL Workstation software.

Acetic acid, propionic acid and butyric acid were purchased from Sigma-Aldrich (Saint Louis, MO, USA) as well as bovine serum albumin fatty-acid free (BSA), human insulin solution, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), Triton X-100 (TX-100), β-nicotinamide adenine dinucleotide reduced disodium salt hydrate (NADH), sodium pyruvate, β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH), sulfosalicylic acid solution (SSA), EthylenediaminetetraAcetic acid tetrasodium salt dihydrate (EDTA), reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase (GR), 2-vinylpyridine (2-VP), 5,5ʹ-Dithio-bis-(2-nitrobenzoic Acid) (DTNB), Potassium dihydrogen phosphate (KH₂PO₄), Dipotassium hydrogen phosphate (K₂HPO₄), human insulin solution, metformin, and menadione. Ethanol and the Pierce bicinchoninic acid (BCA) protein assay kit were purchased from ThermoFisher Scientific (Fremont, CA, USA). The Glucose Uptake-Glo assay kit was purchased from Promega (Leiden, The Netherlands).

Reduced glutathione (GSH) level was assessed using Sigma’s Glutathione Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) after the proteins from the samples were removed by precipitation with a 5% sulfosalicylic acid solution (Sigma-Aldrich) (1:1) followed by 10 min centrifugation at 10,000 rpm and 4 °C. The principle of this colorimetric method consists in the oxidation of GSH by the sulfhydryl reagent 5,5′-dithio-bis (2-nitrobenzoic acid) (DTNB) which leads to the formation of the yellow derivative 5′-thio-2-nitrobenzoic acid (TNB) with a maximum absorption at 412 nm. Finally, GSH levels were calculated as nmols/mg protein by reference to the protein content determined using the Lowry method.