Chloroform

PEO-b-P4VP with the number average molecular weights of PEO and P4VP blocks of 5000 g/mol and 7200 g/mol, respectively, was purchased from Polymer Source Inc. (Montreal, QC, Canada) This sample was denoted as EO4VP. A series of P4VP homopolymers with different molecular weights were also acquired from Polymer Source Inc. Table 1 tabulates the molecular characteristics of the copolymer and homopolymer samples used here. The h-P4VP samples with the number average molecular weights of 1000, 1700, 2300, and 4300 g/mol are denoted as h-P4VP1, h-P4VP2, h-P4VP3, and h-P4VP4, respectively. Chloroform purchased from TEDIA Inc. (Fairfield, OH, USA.) was used as the solvent for preparing the blends.

Standards for PGE₂ (99.0%), PGD₂ (99.0%), PGF_2α (99%), TXB₂ (99%) and PGB₂-d₄ (99.0%) were purchased from Cayman (Ann Arbor, MI, USA). Absolute ethanol, n-hexane, acetonitrile, and formic acid were purchased from J.T. Baker (Deventer, The Netherlands). Ethyl acetate and chloroform were purchased from Tedia (Fairfield, OH, USA). Dexamethasone and LPS from Escherichia coli were purchased from Sigma-Aldrich (St. Louis, MO, USA). Ultrapure water was obtained using a Milli-Q System (Millipore Corporation, Bedford, MA, USA). High glucose Dulbecco’s modified Eagle’s medium (DMEM) without sodium bicarbonate was purchased from HiMedia (Mumbai, India).

Acetonitrile, methanol and isopropanol (HPLC grade) were purchased from Merck (Darmstadt, Germany). Chloroform, formic acid, ammonium acetate and other solvents (analytical grade) were purchased from Tedia (Fairfield, CT, USA). Internal standard (L-2-chlorophenylalanine) was purchased from Sigma Aldrich (St. Louis, MO, USA).

The lipids used: 1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1, 2-dipalmitoyl-3-trimethylammonium-propane (chloride salt) (DPTAP), 1, 2-dipalmitoyl-sn-glycero-3-phospho-(1’-rac-glycerol) (sodium salt) (DPPG), Rhodamine labeled phosphatidylethanolamine (Rho-PE) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Cholesterol was purchased from Sigma-Aldrich, USA. Polycarbonate filter membrane of size 0.2 μm, 0.08 μm and drain discs were purchased from Northern Lipids Inc., Canada. The water used in all the experiments was from a Mili-Q purification system with a resistivity of at least 18.2 ± 0.2 mΩ.cm. The solvents methanol and chloroform were of high-performance liquid chromatography (HPLC) grade (>99% purity) and were purchased from Tedia, USA. Phosphate Buffered Saline (PBS) was prepared from tablets purchased from Sigma-Aldrich, USA. Ranibizumab (Lucentis 10 mg/ml, Novartis AG, Switzerland) were kindly provided by our clinical collaborators and stored at 4 °C in the dark.

For propagation of phage, 10 ml of phage stock was added to 100 ml of V. alginolyticus (3 × 10⁸ CFU ml⁻¹) cultured in MSWYE and incubated in a shaker at 25°C for 3–5 hours until the lysate was clear. The remaining cells and debris were removed by two centrifugation cycles at 10,000 × g for 30 minutes. The supernatant, with a titer of 2 × 10¹⁰ PFU ml⁻¹, was stored at 4°C as a phage stock. To concentrate phages using a standard PEG protocol [7 (link), 20 ], solid NaCl and polyethylene glycol 8000 (Fluka, Germany) were added, and precipitation was performed overnight at 4°C. After centrifugation, the phage particles were resuspended in 2 ml of SM buffer and treated with DNase I and RNase A (Sigma, USA) to remove contaminating nucleic acids from the host. The polyethylene glycol was extracted by adding an equal volume of chloroform (TEDIA, USA) until the interface was clear. The aqueous phase containing the phage was treated with proteinase K (Invitrogen, USA) and sodium dodecyl sulfate (SDS; MD Bio, Inc., USA) at 56°C for 1 h. Phenol extraction was carried out three times at room temperature, and the aqueous phase was further extracted with a 1:1 mixture of equilibrated Phenol (Sigma, USA) and chloroform. DNA precipitated by a 2x volume of cold ethanol was redissolved in deionized water.

The experiment was conducted using one-, two- and three-year-old basin-cultured D. officinale and D. huoshanense seedlings (Fig 1) grown in a greenhouse (Hefei Anhui Mulong Mountain Dendrobium Biotechnology Development Co., Ltd) under the conditions of day 24°C and night 18°C, with natural light. Six replicates of each sample, including one- to three-year-old stems of the two species, were collected from the same pot. Surface soil was removed by washing with water, and the materials were dried with filter paper. Then, the samples were immediately frozen in liquid nitrogen and stored at -80°C until use for metabolomics analysis.
Methanol and chloroform (HPLC grade) were purchased from TEDIA (Fairfield, OH, USA). Pyridine was obtained from Dr. Ehrenstorfer (Augsburg, Germany). Adonitol and methoxylamine hydrochloride were purchased from Sigma-Aldrich. N,O-bis (trimethylsilyl)-trifluoroacetamide (BSTFA) containing 1% trimethylchlorosilane (TMCS) was purchased from SUPELO (Bellefonte, PA, USA). Ultra-pure water was obtained from Wahaha Group Co., Ltd. (Hangzhou, China).