Centrivap concentrator

The HPLC samples were washed using refrigerated CentriVap concentrators purchased from Labconco, Kansas City, MO, USA. The Shimadzu, Kyoto, Japan, UPLC-MS/MS system used for the HPLC studies consisted of an autosampler (SIL-40AC), two solvent feed pumps with a gradient system (LC-40AD), a degasser (DGU- 30A5), a column oven (CTO-40AC), a UV detector (SPD-M20A), and a triple quadrupole mass spectrometer detector (model: LCMS-8045).
A model KJO-4282 Guard Cartridge System and a Lux Cellulose-2 (LC-2) chiral column with a cellulose tris(3-chloro-4-methylphenylcarbamate) stationary phase, as well as a Lux Cellulose-3 (LC-3) chiral column with a cellulose tris(4-methyl benzoate) from Phenomenex Co., Torrance, CA, USA, were used in the chiral chromatographic separations.
All incubations were performed in a dedicated incubator, model Incubators 1000 and Unimax 1010. The incubators were purchased from Heidolph, Schwabach, Germany with controlled temperature and rotation (250 RPM). Each piece of glass used was dried in an oven overnight before being cooled with a stream of nitrogen.

DNA was extracted using a DNeasy^® PowerSoil^® extraction kit (Qiagen, Hilde, Germany) according to the manufacturer’s guidelines. The extracted DNA concentrations were quantified using a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA) and diluted to ~25 ng µL⁻¹ before drying in a CentriVap Concentrator (Labconco, Kansas City, MO, USA). Dried DNA was then shipped at room temperature for sequencing.

Cultures grown in LGIP medium with or without L-tryptophan were analyzed by HPLC to identify and quantify the indolic compounds produced. Samples of 20 mL were collected at the exponential (16 h) and stationary (60 h) phases and centrifuged (8,228 × g; 4°C; 10 min). Cell-free supernatants were loaded in a solid phase cartridge (Strata-X), which was previously activated with methanol and equilibrated with 0.1 M phosphate buffer pH 7.0. Indolic compounds were eluted from cartridge with 10 mL of methanol, which was removed by evaporation in vacuo (Centrivap concentrator, Labconco) at 37°C. The compounds were dissolved in 1 mL of methanol and 50 μL was analyzed using a LC10-A manifold (Shimadzu) and a Luna C18 column (30 cm × 3.9 mm, 5 μm, 100 Å, Phenomenex). The mobile phase was a gradient of phosphate buffer (pH 7.0) and methanol (20–60%) for 40 min pumped at flow rate of 0.5 mL/min. Compounds were detected by ultraviolet absorbance at 254 nm. Identification and quantification was based, respectively, on retention time and the calibration curve of the following compounds: tryptophan, indole-3-acetate, indole-3-lactate (ILA), IAM, IPyA, IAN, tryptamine, anthranilate, indole-3-propionate, indole-3-ethanol, and indole (MP Biomedicals and Sigma Aldrich).

Germline DNA was extracted from patient blood samples (n = 69) and tumor DNA was collected from tumor scrolls (BT collections) or by LMD (ET cell populations) (n = 69). Samples were collected directly into microfuge tubes supplemented with ATL buffer (Qiagen Sciences, LLC, Germantown, MD). Samples were normalized to 360 µL ATL buffer and 40 µL of proteinase K was added for lysis and incubated at 56 °C for 4 h with intermittent shaking. DNA isolation was performed according to the manufacturer’s protocol (DNA Purification from Tissues) using the QiAamp DNA Mini Kit (Qiagen Sciences, LLC). DNA was eluted after a 10 min incubation with 40 µL of Buffer AE, followed by another 10 min incubation with 160 µL of nuclease-free water (Thermo Fisher Scientific) and reduced to 50 µL by vacuum centrifugation (CentriVap Concentrator, Labconco, Kansas City, MO). Quantity and purity (260/280 ratio) were assessed spectrophotometrically (Nanodrop 2000 Spectrophotometer, Thermo Fisher Scientific, Inc.) and fluorometrically (Quant-iT PicoGreen dsDNA Assay Kit, Thermo Fisher Scientific) according to manufacturer’s protocols.

To determine the concentration of ethanol-solved compounds, the solvent was evaporated under vacuum conditions (CentriVap Concentrator, Labconco) from a known volume of the extracts and the concentration (w/v) was calculated by weighting the mass of solid ethanol-solved compounds on an analytical balance (PRL TA 13, MERA-WAG, Gdansk, Poland). The extracts were adjusted to a concentration of 3 mg/mL and used for analytical tests 4.4–4.6.

According to the related reference [35 (link)], the samples were taken at the specific nodes and serum samples were prepared. Above 3 mL, venous blood was gotten from specific rabbits. The blood samples were centrifuged for 10 min (4 °C, 3500 r/min) to collect the serum sample. On the analyzing day, after serum sample thawed, vortex 3 min by using Vortex Mixer XW-80A. Then, 100 μL serum sample was taken for metabolomics analysis. The proteins in sample were removed by using 400 μL acetonitrile. The supernatant was evaporated to dryness by Centrivap Concentrator (LABCONCO, USA). The residue was reconstituted in the initial mobile phase, vortex 3 min, and ultrasonic extracted 5 min at 4 °C. Through the centrifugation, supernatant was transferred into 150 μL glass insert in a 1.5 mL amber glass vial and analyzed.

Plasma samples were thawed at room temperature before preparation. To precipitate protein, 200 μL plasma samples were extracted with 600 μL methanol, and the mixture was then vortexed for 1 min and centrifuged at 3000 rpm for 10 min. Afterward, 400 μL supernatants were transferred into new centrifuge tubes, and were evaporated to dryness at 37°C by using Labconco CentriVap concentrator (Kansas City, MO, United States). The residues were re-dissolved with 100 μL of initial mobile phase, vortexed for 3 min and centrifuged at 13000 rpm for 10 min at 4°C. Finally, an aliquot of 5 μL supernatant was injected for UHPLC-MS analysis.
Furthermore, 15 plasma samples were randomly selected from these three groups, which were mixed together as quality control (QC) samples. As QC samples contained the most data of each group, it was applied to validate the stability of UHPLC-MS system. Before analysis, QC samples were analyzed for six times to equilibrate the UHPLC system, then every ten samples were used to monitor the stability of this method (Godzien et al., 2011 (link); Prosser et al., 2014 (link)). All samples above were maintained at 4°C during analysis.