Hp agilent 1200

C57Bl/6 mice were inoculated subcutaneously with 10⁶ GL261-WT or GL261-hIDO1 cells in 100 mL PBS into the left flank. Plasma and tumours were collected from euthanised mice at 14, 17 and 21 days post inoculation (n = 6 per group) and analysed for concentration of kynurenine and tryptophan. In brief, 100µL of each plasma sample was mixed with 100µL of potassium phosphate buffer (0.05 M, pH 6) containing 100µM of the internal standard, 3-Nitro-L-Tyrosine (Sigma-Aldred, St Louis, MO, USA). Twenty-five µL of 2 mol/L trichloroacetic acid (Merck) was added to precipitate the proteins, and the samples were then vortexed and centrifuged at 12,000× g for 6 min, at room temperature. Standard calibration curves made from serial dilution of tryptophan (Sigma) and L-kynurenine (Sigma) were prepared with the same treatment as the samples. All supernatants collected after centrifugation were transferred into micro sampling vials and analysed for kynurenine and tryptophan as previously described [43 (link)] by HPLC (HP Agilent 1200, Agilent Technologies, Walbronn, Germany). Kynurenine and tryptophan concentrations in samples were calculated from the standard calibration curves and the kynurenine to tryptophan ratio (K:T) was calculated by dividing the concentration of kynurenine by the tryptophan concentration.

Analytical HPLC was used to assess the purity of the extract during each step of the isolation procedure. The instrument used was an HP Agilent 1200 analytical HPLC system (HP/Agilent, Santa Clara, USA) equipped with a diode array detector at 520 nm and 280 nm, and a 250 × 25 mm (4.5 µm particle size) Ascentis RP-Amide column (Supelco, Bellefonte, USA. The mobile phases were A, 1% TFA in water (v/v), and B, 1% TFA in acetonitrile (v/v), used at a flow of 1 mL/min. A solvent elution profile consisted of initial conditions of 30% B and the following isocratic and gradient elution: 0–7 min gradient to 40% B, 7–10 min gradient to 50% B, 10–15 min isocratic, 15–17 min gradient to 80% B, 17–22 min isocratic, and 22–25 min gradient to 30%. Injections were 20 µL aliquots injected by an auto-sampler. Prior to injections, samples were filtered through a 0.45 µm filter.