Milli q grade water

Baby Hamster Syrian Kidney (BHK-21cells) were cultured in Dulbeccoʼs modified Eagleʼs medium (DMEM) supplemented with 10% FBS. FMDV strains, O/MYA98/BY/2010 and Asia1/JSL/ZK/06 investigated extensively as vaccine strains, were provided by Zhongnongweite biotechnology Co., Ltd. Each strain was propagated in BHK-21 cells at a multiplicity of infection of 0.001 and incubated in 5% CO₂ at 37 °C. A PrimeScriptTM RT reagent kit containing gDNA Eraser and SYBR Premix Ex TaqTM II (Tli RNaseH Plus) was purchased from TaKaRa (Dalian, China). MTS assay was available from Abcam (Cambridge, UK). The 50% tissue culture infectious dose (TCID₅₀) was measured with the Reed and Muench method. Trehalose, CuSO₄·5H₂O were purchased from sigma (MO, USA). All other chemicals were analytical grade reagents purchased from Beijing Chemical works (Beijing, China) and all solutions were prepared using Milli-Q grade water (Millipore,USA).

Agarose was purchased from Lonza (USA), Promega (USA) and Biowest (Spain), respectively. Blue Dextran (2000 kDa), thyroglobulin (660 kDa), BSA (66.12 kDa), RNA (13.7 kDa), and fluorescein isothiocyanate (FITC) were from Sigma (USA). rHBSAg were kindly provided by National Engineering Research Center for Biotechnology (China). QZT 4FF was kindly provided by Senhui Microspheres Tech (Suzhou) (Suzhou, Jiangsu, China). All other chemicals were of analytical grade and all solutions were made using Milli Q‐grade water (Millipore, USA).

Coffea arabica beans (wet processed, screen size ≥15, zero primary and secondary defects, high aromatic profile, clean cup) were sourced from Brazil and roasted at temperature up to 220°C under industrial conditions for durations of 2, 4, 6, 8, 10 and 12 minutes. Samples (25 g for each roasting time) were immediately sealed under inert atmosphere in individual foil bags prior to shipping and further analysis. Australian quarantine restrictions prevented us from importing unprocessed green coffee beans. Upon receipt, the foil bags were opened and the beans were studied either in-tact or following grinding. Coarse-ground samples were prepared by crushing ca. 10–20 beans with a marble mortar and pestle. To assist grinding, 2 min roasted beans were first frozen in liquid nitrogen.
To prepare coffee brews, 3.5 g of 12 min-roasted beans was coarse ground. Within 15 min of grinding, 20 mL ‘MilliQ’ grade water (Millipore) was added and brewed at 92 ± 1°C in a beaker on a heat block with constant stirring. After 5 min, the solutions were rapidly cooled on ice and clarified using a low-binding Millex 0.45 μm PVDF syringe filter (Millipore). The resulting solutions were either used immediately or stored at—80°C prior to further use.

Low molecular weight chitosan (120 kDa; deacetylation degree of 90–95%, Sigma Aldrich^®, St. Louis, MO, USA), hypromellose phthalate (75 kDa; phytalyl content of 31%, Shin-Etsu^®, Tokyo, Japan), Müeller Hinton agar plus 5% sheep blood; Müeller Hinton broth supplemented with 50% fetal bovine serum; Helicobacter pylori bacterial strain (ATCC 4354); metronidazole (Henrifarma^®, São Paulo, Spain); mucine type II (Sigma Aldrich^®); resazurin (Sigma Aldrich^®). All the other materials used were of analytical grade and obtained from commercial suppliers. Milli-Q grade water (Millipore, Molsheim, France) was used for sample preparation and the assays.

Oxaliplatin powder (Actavis New Zealand) was dissolved in 5% glucose solution, sonicated and filtered with a 0.22 μm Millipore filtration system (Bedford, USA) and stored at -20°C. The membrane vesicle incubation buffer solution consisted of 50 mM MOPS-Tris, 70 mM KCl, 7.5 mM MgCl₂ and was freshly prepared using MOPS-Tris, KCl and MgCl₂ powders dissolved in Milli-Q grade water (Millipore, Bedford, USA). All other chemicals were sourced from Sigma-Aldrich, St Louis, MO, USA. Stock solutions of 5(6)-carboxy-2,'7'-dichlorofluorescein (CDCF), myricetin and MK571 were prepared at concentrations 1000 times higher than working solutions in dimethylformamide and stored at -20°C.

Reference standards of htyr [3,4-Dihydroxyphenethyl alcohol], tyr [2-(4-Hydroxyphenyl) ethanol], and ole [2-(4-Hydroxyphenyl) ethyl (3S,4E)-4-formyl-3-(2-oxoethyl) hex- 4-enoate] were acquired from Sigma-Aldrich (St. Louis, MO). These compounds were dissolved in methanol and preserved at −20 °C for utilization in the solutions under study. Analytical grade solvents or HPLC (Sigma-Aldrich), and Milli-Q grade water (Millipore Corp, Bedford, MA, USA) were used.

All buffers and solutions were
made using Milli-Q grade water with 18.2 MΩ resistivity (Millipore,
USA). Chemicals were purchased from Sigma-Aldrich unless otherwise
indicated.