Waters 600 hplc

Primary analytical instruments and chemicals used were as follows: Waters 600 HPLC (Waters, MA, USA) with an L-column2 ODS column (250 mm × i.d. 4.6 mm; particle size 5 μm); Agilent 218 Purification Systems (Agilent, Santa Clara, CA, USA) with a Prep-C18 column (50 mm × i.d. 30 mm; particle size 5 μm); Agilent 7890A GC (Agilent, CA, USA) with an HP-5 capillary column (30 m × i.d. 0.25 mm); MS spectrometers JEOL JMS-T100GCV, JMS-SX-102 (JEOL, Tokyo, Japan) and Agilent 7000C GC-MS/MS (Santa Clara, CA, USA) with an Agilent HP-5 glass capillary column (Agilent Technologies, 30 m × i.d. 0.32 mm) under the conditions of initial temperature of 100 °C for 1.5 min and heating at a rate of 60 °C min⁻¹ until 300 °C with a carrier gas of He; NMR spectrometers JEOL JNM-EX270 (JEOL, Tokyo, Japan) and Bruker AM 500 (Bruker, Bremen, Germany); ABI Prism 310 Genetic Analyzer (Applied Biosystems, CA, USA); StepOnePlus Real-Time PCR thermal cycling block (Applied Biosystems, CA, USA); trifluoromethanesulfonic acid-d (TfOD, Energy Chemical, Shanghai, China); L-phenylalanine (TCI, Tokyo, Japan); L-phenylalanine-[ring-²H₅] (Sigma-Aldrich, MO, USA); phenylacetic acid (PAA, Sigma-Aldrich, St. Luis, MO, USA). Other chemicals used for preliminary screenings in the present study were purchased from TCI and Wako (Osaka, Japan).

The chromatographic conditions for the detection of sugars were as follows. Waters-600 HPLC was used with a Sugarpak I column at a column temperature of 85 °C, using the evaporative light scattering detection method. The mobile phase was ultrapure water, and the flow rate was 0.4 mL/min.

In order to determine the identity of molecules from CM bound to tissue culture plastic, two different extraction methods were used to remove the molecules from the plastic. First, after overnight incubation of wells of a 24‐well tissue culture plate were with 30× concentrated lung epithelial CM, bound protein was extracted using 1% sodium lauryl sulfate (SDS) and 0.2% deoxycholate buffer. Scattering assays of duplicate wells that were not extracted confirmed that the migration‐inducing substances were bound to the plastic. MCF‐7 cells added to the wells after extraction no longer exhibited scattering, confirming the removal of motility‐inducing substances. SDS‐deoxycholate‐extracted samples were supplemented with SDS to 4% and then subjected to filter‐assisted sample preparation (FASP) 28. Peptides trypsinized from the FASP membrane (1:50 trypsin:substrate) were isolated using C18 STAGE‐tips 29. Eluted, vacuum‐dried peptides were redissolved in 10 μL of 0.1% formic acid in water for nanoLC‐MS/MS, using an LTQ mass spectrometer (Thermo‐Fisher) with Waters 600 HPLC running with split flow. The five most intense precursor peptides per spectrum were subjected to fragmentation and, using Mascot 2.3, the resulting peaklist against a SwissProt database with human taxonomy was searched. In addition, a decoy search was performed using a database of common contaminants.

The biomass was determined by measuring the dry weight of shoots and roots. After the treatments, the fresh tissues were dried at 80°C until the weight was constant, and then cooled in the dryer.
The dry plant tissues were mixed with HNO₃ and HClO₄ (v/v, 83/17) for 24 h. The mixtures were then digested at 90°C for 3 h, 150°C for 5 h, and 180°C until nearly dry. The digested products were cooled and dissolved in ddH₂O to reach a total volume of 25 mL. The Cd concentrations were measured using an atomic absorption spectrophotometer.
To estimate the sugar, the shoot and root samples were dried at 105°C for 30 min, and at 70°C for 24 h. They were then homogenized in 80% ethanol, boiled at 70°C for 30 min, and centrifuged at 8000 g at 4°C for 10 min. The total soluble sugars in the supernatants were measured using the method of McCready et al. [41 ]. The fructose, glucose, and sucrose contents were measured using high performance liquid chromatography (HPLC, Waters 600 HPLC), as described by Sánchez-Linares Luis et al. [42 ].